Need-to-Know NAFLD: The Complete Guide to Nonalcoholic Fatty Liver Disease (by Team-IRA) [1 ed.] 1032479493, 9781032479491

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Need-to-Know NAFLD Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the obesity and metabolic syndrome epidemics, which this up-to-date book deals with comprehensively. The contents outline disease mechanisms, diagnostic tests, management, varying manifestations and special populations. It covers the mechanistic pathways that contribute to NAFLD development, including the role of genetic variants and the gut microbiome. It elaborates on noninvasive diagnostic tests to screen for NAFLD, determine its severity, and monitor response to lifestyle intervention and pharmacologic treatment. This book helps clinicians to diagnose and treat this common and potentially deadly disease. Key Features: ■

Reviews current drugs in development and provides practical advice to clinicians on the diagnosis and management of fatty liver.

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Proves attractive to primary care providers who are on the front line of managing patients with NAFLD, to gastroenterologists and hepatologists who would beneft from updated data on how to risk-stratify patients and identify those who will be eligible for pharmacologic treatment, and other specialists such as cardiologists, endocrinologists and nephrologists who will fnd this book to be a useful reference on the extrahepatic manifestations of NAFLD.

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Focuses on extrahepatic manifestations and new insights on the mechanistic drivers of the disease.

Need-to-Know NAFLD The Complete Guide to Nonalcoholic Fatty Liver Disease

Edited by

Naim Alkhouri and Stephen A. Harrison

Designed cover image: Editors First edition published 2024 by CRC Press 6000 Broken Sound Parkway NW, Suite 300, Boca Raton, FL 33487–2742 and by CRC Press 4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN CRC Press is an imprint of Taylor & Francis Group, LLC © 2024 selection and editorial matter, Naim Alkhouri and Stephen A. Harrison; individual chapters, the contributors This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily refect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientifc or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verifed. The reader is strongly urged to consult the relevant national drug formulary and the drug companies’ and device or material manufacturers’ printed instructions, and their websites, before administering or utilizing any of the drugs, devices or materials mentioned in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under US Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microflming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978–750–8400. For works that are not available on CCC please contact [email protected] Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identifcation and explanation without intent to infringe. ISBN: 978-1-032-47949-1 (hbk) ISBN: 978-1-032-47948-4 (pbk) ISBN: 978-1-003-38669-8 (ebk) DOI: 10.1201/9781003386698 Typeset in Palatino by Apex CoVantage, LLC

To my two daughters, Giada and Tala: you are the light that guides me in my journey through life. I hope that you fnd me to be a cool dad one day. Naim Alkhouri This book is dedicated to my loving wife, Renee, and my two amazing children, Taylor and Anna-Lauren. Your love and support have been amazing. I also want to thank the many patients with NAFLD/NASH who have given me hope and inspiration to push forward and continue to advance the feld. Stephen A. Harrison

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Naim Alkhouri and Stephen A. Harrison Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Section I: Mechanisms of NAFLD Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1. Historical Perspectives and Clinical Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Imad Asaad and Naim Alkhouri 2. Epidemiology and Natural History of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Linda Henry and Zobair M. Younossi 3. Genetics of NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Luca Valenti and Cristiana Bianco 4. Mechanisms of Hepatocyte Injury and Infammation in NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 Gopanandan Parthasarathy and Harmeet Malhi 5. Role of the Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 Carlos J. Pirola and Silvia Sookoian Section II: Diagnostic Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 6. Simple Algorithms in Primary Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Xixi Xu and Michelle T. Long 7. Ultrasound-Based Techniques in NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Vikas Taneja, Nezam H. Afdhal and Michelle J. Lai 8. MRI-Based Technologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Victor de Lédinghen 9. Artifcial Intelligence in NAFLD Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Joseph C. Ahn and Samer Gawrieh Section III: Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 10. Dietary Interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Monica Tincopa 11. Weight Loss Medications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Mohammad Qasim Khan, Manhal Izzy and Kymberly D. Watt 12. De Novo Lipogenesis Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Brent A. Neuschwander-Tetri 13. Targeting Bile Acids (FXRs and FGF19) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Daniel Garrido, Rukaiya Bashir Hamidu and Dina Halegoua-DeMarzio 14. PPAR Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Sven M. A. Francque 15. Anti-Infammatory Drugs: Metabolic Infammation in NASH—A Drug Target? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 Angelo Armandi and Jörn M. Schattenberg 16. Practical Aspects of Pharmacologic Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Naim Alkhouri and Stephen A. Harrison

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CONTENTS

Section IV: Extrahepatic Manifestations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 17. Endocrinology: Diabetes and Other Endocrinopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 Scott Isaacs and Julio Leey 18. Obstructive Sleep Apnea and Nonalcoholic Fatty Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Dania Brigham and Shikha S. Sundaram 19. Extrahepatic Gastrointestinal Manifestations of Nonalcoholic Fatty Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Rinjal Brahmbhatt and Mousab Tabbaa 20. Extrahepatic and Hepatic Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Maryam Ibrahim and Tracey G. Simon Section V: NAFLD in Special Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 21. NAFLD in Children: Unique Aspects and Controversies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 Samar H. Ibrahim and Rohit Kohli 22. NAFLD in HIV Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Giada Sebastiani 23. NAFLD in Liver Transplant Recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Liyun Yuan and Norah Terrault 24. NAFLD in Lean Individuals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Donghee Kim and Vincent Wai-Sun Wong Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

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Preface Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the obesity and metabolic syndrome epidemics. It is the most common form of chronic liver disease, affecting 25% of the global population. NAFLD has become a leading cause for cirrhosis, liver cancer, and the need for liver transplantation. It also contributes signifcantly to the development of cardiovascular disease, diabetes and chronic kidney disease. Our understanding of mechanistic pathways that contribute to NAFLD development and progression has expanded exponentially, including the role for genetic variants and the gut microbiome. Several advancements have been made over the past decade in noninvasive diagnostic tests to screen for NAFLD, determine its severity, and monitor response to lifestyle intervention and pharmacologic treatment.

Several agents are in phase 3 clinical trials with expected FDA approval by 2023 providing a rationale for a reference book that clinicians can use to diagnose and treat this common and potentially deadly disease. We hope that this book will be attractive to primary care providers who are on the front line of managing patients with NAFLD. Gastroenterologists and hepatologists will fnd this book of interest in terms of providing the most updated data on how to risk-stratify patients and identify those who will be eligible for pharmacologic treatment. Other specialists such as cardiologists, endocrinologists and nephrologists will fnd this book to be a useful reference on the extrahepatic manifestations of NAFLD. Naim Alkhouri and Stephen A. Harrison

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Editors Naim Alkhouri, MD, is the Chief Medical Offcer (CMO), Chief of Transplant Hepatology, and Director of the Fatty Liver Program at Arizona Liver Health (ALH) in Phoenix, Arizona. Prior to joining ALH, Dr. Alkhouri served as the director of the Metabolic Health Center at the Texas Liver Institute and Associate Professor of Medicine and Pediatrics at the University of Texas (UT) Health in San Antonio, Texas. Dr. Alkhouri completed his Gastroenterology and Transplant Hepatology training at the renowned Cleveland Clinic in Cleveland, Ohio, where he was also appointed Assistant Professor of Medicine and Director of the Metabolic Liver Disease Clinic at the Cleveland Clinic Digestive Disease and Surgery Institute. Dr. Alkhouri is a key opinion leader in the feld of NASH therapeutics and an advisor/consultant to many pharmaceutical and biomarker development companies. He is Principal Investigator on several multicenter global NASH trials and a member of the AASLD NASH Special Interest Group (NASH SIG). Dr. Alkhouri has been published in more than 200 publications including the New England Journal of Medicine, Lancet, JAMA, Gastroenterology, Hepatology, and Journal of Hepatology. He presents his work at both national and international medical conferences. Among many research awards, Dr. Alkhouri received the American College of Gastroenterology Junior Faculty Development Award to study the analysis of breath volatile organic compounds to diagnose nonalcoholic fatty liver disease.

Stephen A. Harrison, MD, is the Founder and Chairman of Pinnacle Clinical Research and Cofounder and Chairman of Summit Clinical Research, LLC in San Antonio, Texas. He is board-certifed in Internal Medicine and Gastroenterology. Dr. Harrison earned his medical degree from the University of Mississippi School of Medicine. He completed his internal medicine residency and gastroenterology fellowship at Brooke Army Medical Center before completing a 4-year advanced liver disease fellowship at Saint Louis University. Dr. Harrison served as a Professor of Medicine at the Uniformed Services University of the Health Sciences and is currently a Visiting Professor of Hepatology at Radcliffe Department of Medicine, University of Oxford. Dr. Harrison served as a colonel in the United States Army. Retiring in 2016, he concluded 20 years of dedicated service to his country. During his army tenure, he served as the Director of Graduate Medical Education at Brooke Army Medical Center, Associate Dean for the San Antonio Uniformed Services Health Education Consortium and Gastroenterology Consultant to the Army Surgeon General. He is a past Associate Editor for Hepatology and Alimentary Pharmacology & Therapeutics journals. He is internationally known for studies in nonalcoholic fatty liver disease (NAFLD) with more than 300 peer-reviewed publications in top-tier journals including the New England Journal of Medicine, Nature Medicine, Lancet, JAMA, Gastroenterology, Journal of Hepatology, and Hepatology. He has an H-Index of 93.

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Contributors Nezam H. Afdhal Division of Gastroenterology and Hepatology Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts, USA  Joseph C. Ahn Division of Gastroenterology and Hepatology Mayo Clinic Rochester, Minnesota, USA  Angelo Armandi Metabolic Liver Disease Research Program I. Department of Medicine University Medical Center of the Johannes Gutenberg–University Mainz, Germany Division of Gastroenterology and Hepatology Department of Medical Sciences University of Turin Turin, Italy  Imad Asaad Cleveland Clinic Foundation Cleveland, Ohio, USA Cristiana Bianco Precision Medicine–Department of Transfusion Medicine and Hematology Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milan, Italy  Rinjal Brahmbhatt North Shore Gastroenterology & Endoscopy Centers Westlake, Ohio, USA  Dania Brigham Children’s Hospital Colorado University of Colorado Hospital Aurora, Colorado, USA  Sven M. A. Francque Department of Gastroenterology and Hepatology Antwerp University Hospital Antwerp, Belgium Laboratory of Experimental Medicine and Paediatrics (LEMP) Faculty of Medicine and Health Sciences University of Antwerp Antwerp, Belgium InfaMed Centre of Excellence University of Antwerp Antwerp, Belgium Translational Sciences in Infammation and Immunology University of Antwerp Antwerp, Belgium European Reference Network on Hepatological Diseases (ERN RARE-LIVER) Daniel Garrido Department of Medicine Division of Gastroenterology and Hepatology

Thomas Jefferson University at Sidney Kimmel Medical College Philadelphia, Pennsylvania, USA  Samer Gawrieh Division of Gastroenterology and Hepatology Department of Medicine Indiana University School of Medicine Indianapolis, Indiana, USA Dina Halegoua-DeMarzio Department of Medicine Division of Gastroenterology and Hepatology Thomas Jefferson University at Sidney Kimmel Medical College Philadelphia, Pennsylvania, USA  Rukaiya Bashir Hamidu Department of Medicine Division of Gastroenterology and Hepatology Thomas Jefferson University at Sidney Kimmel Medical College Philadelphia, Pennsylvania, USA  Linda Henry Betty and Guy Beatty Center for Integrated Research Inova Health System Falls Church, Virginia, USA Inova Medicine, Inova Health System Falls Church, Virginia, USA Center for Outcomes Research in Liver Disease Washington, DC, USA  Maryam Ibrahim Department of Medicine Massachusetts General Hospital Boston, Massachusetts, USA Harvard Medical School Boston, Massachusetts, USA Samar H. Ibrahim Mayo Clinic Rochester, Minnesota, USA  Scott Isaacs Emory University School of Medicine Atlanta, Georgia, USA  Manhal Izzy Division of Gastroenterology Hepatology and Nutrition Vanderbilt University Nashville, Tennessee, USA  Mohammad Qasim Khan Division of Gastroenterology University of Western Ontario London, Ontario, Canada  Donghee Kim Division of Gastroenterology and Hepatology Stanford University School of Medicine Stanford, California, USA 

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CONTRIBUTORS

Rohit Kohli Children’s Hospital Los Angeles Los Angeles, California, USA Michelle J. Lai Division of Gastroenterology and Hepatology Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts, USA  Victor de Lédinghen Hepatology and Liver Transplantation Unit CHU Bordeaux and INSERM U1312 Bordeaux University Bordeaux, France  Julio Leey Division of Endocrinology Diabetes and Metabolism University of Florida Gainesville, Florida, USA

Giada Sebastiani Chronic Viral Illness Service McGill University Health Centre Montreal, Québec, Canada Division of Gastroenterology and Hepatology Royal Victoria Hospital McGill University Health Centre Montreal, Québec, Canada  Tracey G. Simon Division of Gastroenterology and Hepatology Massachusetts General Hospital Boston, Massachusetts, USA Clinical and Translational Epidemiology Unit (CTEU) Massachusetts General Hospital Boston, Massachusetts, USA

Harmeet Malhi Division of Gastroenterology and Hepatology Mayo Clinic Rochester, Minnesota, USA 

Silvia Sookoian Systems Biology of Complex Diseases Centro de Altos Estudios en Ciencias Humanas y de la Salud (CAECIHS) Universidad Abierta Interamericana Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET) Buenos Aires, Argentina Clinical and Molecular Hepatology Centro de Altos Estudios en Ciencias Humanas y de la Salud (CAECIHS) Universidad Abierta Interamericana Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET) Buenos Aires, Argentina

Brent A. Neuschwander-Tetri Department of Gastroenterology and Hepatology Saint Louis University St. Louis, Missouri, USA 

Shikha S. Sundaram Children’s Hospital Colorado University of Colorado Hospital Aurora, Colorado, USA 

Gopanandan Parthasarathy Division of Gastroenterology and Hepatology Mayo Clinic Rochester, Minnesota, USA 

Mousab Tabbaa North Shore Gastroenterology & Endoscopy Centers Westlake, Ohio, USA

Michelle T. Long Section of Gastroenterology Department of Medicine Boston University School of Medicine Boston, Massachusetts, USA 

Carlos J. Pirola Systems Biology of Complex Diseases Centro de Altos Estudios en Ciencias Humanas y de la Salud (CAECIHS) Universidad Abierta Interamericana Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET) Buenos Aires, Argentina Clinical and Molecular Hepatology Centro de Altos Estudios en Ciencias Humanas y de la Salud (CAECIHS) Universidad Abierta Interamericana Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET) Buenos Aires, Argentina  Jörn M. Schattenberg Metabolic Liver Disease Research Program I. Department of Medicine University Medical Center of the Johannes Gutenberg–University Mainz, Germany 

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Vikas Taneja Division of Gastroenterology and Hepatology Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts, USA  Norah Terrault Keck Medicine at University of Southern California Los Angeles, California, USA Monica Tincopa Transplant Hepatology Department of Internal Medicine Division of Digestive Diseases University of California Los Angeles Santa Monica, California, USA  Luca Valenti Precision Medicine–Department of Transfusion Medicine and Hematology Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Milan, Italy

CONTRIBUTORS

Department of Pathophysiology and Transplantation Università Degli Studi di Milano Milan, Italy  Kymberly D. Watt Division of Gastroenterology and Hepatology Mayo Clinic Rochester, Minnesota, USA  Vincent Wai-Sun Wong Department of Medicine and Therapeutics The Chinese University of Hong Kong Hong Kong 

Zobair M. Younossi Betty and Guy Beatty Center for Integrated Research Inova Medicine, Inova Health System Falls Church, Virginia, USA Center for Outcomes Research in Liver Disease Washington, DC, USA Center for Liver Disease Department of Medicine Inova Fairfax Medical Campus Fairfax, Virginia, USA  Liyun Yuan Keck Medicine at University of Southern California Los Angeles, California, USA

Xixi Xu Department of Medicine Boston University School of Medicine Boston, Massachusetts, USA 

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SECTION I

MECHANISMS OF NAFLD DEVELOPMENT  

1

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

1 Historical Perspectives and Clinical Presentation Imad Asaad and Naim Alkhouri

CONTENTS 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2 Historical Perspectives of Terminology and Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Historical Perspectives of Pathophysiology, Natural History and Association with Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3.1 Association Between Metabolic Syndrome Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3.2 Association of NAFLD and Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3.3 Apoptosis, Lipid and Bile Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3.4 Infammatory Response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.3.5 Genetics of NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.4 Historical Perspectives of Diagnostic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.5 Historical Perspectives of Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.1 INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is currently the most prominent cause of chronic liver disease worldwide. NAFLD is defned by the presence of hepatic steatosis by imaging or histology, affecting at least 5% of hepatocytes in individuals without secondary causes of hepatic fat accumulation such as signifcant alcohol consumption, viral hepatitis, steatogenic medications (e.g., tamoxifen, amiodarone, methotrexate), or lipodystrophy1. NAFLD can be categorized histologically into nonalcoholic fatty liver (NAFL), the potentially nonprogressive subtype of NAFLD, as well as nonalcoholic steatohepatitis (NASH), the potentially progressive subtype of NAFLD that can lead to advanced fbrosis and cirrhosis, as well as liver-related morbidity and mortality. NASH is defned histologically by the presence of hepatic steatosis with evidence for hepatocyte damage and liver infammation1,2. Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide, with a global prevalence of 25.24%3. In a cross-sectional study from 2011 to 2014, the overall prevalence of NAFLD among US adults was 21.9%, representing 51.6 million individuls4. And NASH-related cirrhosis is currently the second most common indication for liver transplants in the United States5. The prevalence of NAFLD in patients with components of metabolic syndrome is higher. For instance, NAFLD has been reported in over 76% of type 2 diabetics. Furthermore, over 90% of severely obese patients undergoing bariatric surgery have NAFLD6. A high burden of metabolic comorbidities is associated with NAFLD creating implications for clinical management of the disease. Given the common risk factors between NAFLD and cardiovascular disease (CVD), cardiac-related death is one of the leading causes of death for NAFLD patients. Patients with NAFLD have increased overall mortality compared to matched control populations without NAFLD, and patients with histological NASH have an increased liver-related mortality rate7. NAFLD is now considered the third most common cause of hepatocellular carcinoma (HCC) in the United States, likely related to the increasing prevalence of this condition. Extrahepatic cancer-related mortality is among the top three causes of death in subjects with NAFLD1. 2

The signifcant prevalence and burden of the disease have led to major efforts during the last two decades in the development of diagnostics and treatments. However, NAFLD is a heterogeneous disease that has multiple metabolic and genetic factors contributing to its progression. It is largely asymptomatic in the early stages but can lead to signifcant clinical outcomes in the late stages. This variability in NAFLD natural history has led to substantial challenges in biomarkers discovery and drug development. Learning the history of the disease will help provide better understanding about its pathophysiology and natural history, which are the cornerstones in the development of diagnostic testing and therapeutics. The history of NAFLD has multiple major milestones. This chapter will provide an overview of the most important landmark discoveries and studies in the history of NAFLD that have formed our current understanding of this disease. These are the major areas discussed in this chapter: 1. Historical perspectives of terminology and histopathology 2. Historical perspectives of pathophysiology, natural history and the association with metabolic syndrome a. Association between metabolic syndrome components b. Association of NAFLD and metabolic syndrome c. Apoptosis, lipid and bile acid metabolism d. Infammatory response e. Genetics of NAFLD 3. Historical perspectives of diagnostic testing 4. Historical perspectives of therapeutics 1.2 HISTORICAL PERSPECTIVES OF TERMINOLOGY AND HISTOPATHOLOGY In the 1800s, multiple pathologists reported fatty infltration of the liver in individuals with diabetes and obesity similar to those with alcoholic liver disease8–10. In 1962, the link between fat accumulation in the liver and liver injury was frst recognize by Thaler11. DOI: 10.1201/9781003386698-2

1 HISTORICAL PERSPECTIVES AND CLINICAL PRESENTATION

In 1980, Ludwig and colleagues at the Mayo Clinic introduced the term “nonalcoholic steatohepatitis” (NASH) based on histological features. They studied liver biopsies of 20 patients who had hepatitis of unknown cause and did not consume alcohol or consumed alcohol in quantities not considered harmful to the liver. Biopsy specimens had histological fndings similar to alcoholic hepatitis characterized by the presence of fatty changes with evidence of lobular hepatitis, focal necrosis with mixed infammatory infltrates, Mallory bodies and evidence of fbrosis12. In 1983, Moran reported the presence of steatohepatitis in three obese children who presented with nonspecifc abdominal pain and abnormal liver function tests13. Then the term “nonalcoholic fatty liver disease” (NAFLD) was frst introduced by Schaffner and Thaler in 198614. In 2020, multiple experts reached consensus that the terminology “nonalcoholic” overemphasizes “alcohol” and underemphasizes metabolic risk factors. So a name change from NAFLD to metabolic-associated fatty liver disease (MAFLD) has been proposed15. Subsequent studies afterward showed that MAFLD is associated with an increased risk of all-cause mortality and increased risk of cardiovascular disease16,17. However, this proposal was considered by other experts to be premature and suggested that a change will be justifed when a more scientifc and complete understanding of the disease pathogenesis, risk stratifcation, molecular phenotyping and related therapeutic approaches are elucidated18. 1.3 HISTORICAL PERSPECTIVES OF PATHOPHYSIOLOGY, NATURAL HISTORY AND ASSOCIATION WITH METABOLIC SYNDROME 1.3.1 Association between Metabolic Syndrome Components In 1765, J.B. Morgagni was the frst to identify the association between components of metabolic syndrome. He clearly described the association between visceral obesity, hypertension, hyperuricemia, atherosclerosis and obstructive sleep apnea long before the modern recognition of this syndrome19. In the early twentieth century, obesity incidence was increasing and accompanied by a rising incidence of diabetes. Elliott Joslin was the frst US doctor to specialize in diabetes. In 1924, Joslin noted: “Diabetes is 15 times as common among adults and 20 times as common among the fat”20. In 1939, Harold Himsworth identifed the two different types of diabetes: The insulin-sensitive type, which is what we call now type 1, and the insulin-insensitive type, which is what we call now type 2. Himsworth described that insensitive diabetics tend to be elderly and obese and to have hypertension and arteriosclerosis20. Then multiple European scientists in 1900s described the very common coexistence of the various components of the syndrome, including hypertension obesity, hypertension and hyperlipidemia21–23. In 1988, Raevan introduced the term “Syndrome X” and further described the role of insulin resistance in the development and progression of T2D, HTN and CAD24. 1.3.2 Association of NAFLD and Metabolic Syndrome In 1932, Zelman described the presence of liver damage in obesity25. Then in 1970, Beringer and Thaler reported

NAFLD association with obesity and diabetes in a study including 465 liver biopsies of diabetic patients26. In 1979, Itoh reported fve cases of nonalcoholic diabetic women with obesity and hyperglycemia who had histological fndings consistent with micronodular cirrhosis27. In 1980, study by Ludwig et al. that introduced the term NASH, most of the study’s 20 patients were women with obesity and type 2 diabetes12. In 1990, Elizabeth Powell and colleagues identifed obesity, hyperlipidemia and T2DM as risk factors for nonalcoholic steatohepatitis. In this study, 42 patients with nonalcoholic steatohepatitis were followed for a median of 4.5 years (range = 1.5–21.5 years). Except for two patients with lipodystrophy, all were obese; 35 of 42 were women, 26 of 32 were hyperlipidemic and 15 were hyperglycemic28,29. In 1994, Bruce Bacan analyzed a series of 33 patients with NASH. He noted in his study that NASH spectrum should be expanded as compared to Ludwig’s initial description and should no longer be considered a disease predominantly seen in obese women with diabetes30. A study by Marchesini and colleagues in 1999 reported the association between nonalcoholic fatty liver disease and insulin resistance even in the absence of diabetes and obesity31. Another study by Aron Sanyal and colleagues indicated that peripheral insulin resistance, increased fatty acid beta-oxidation and hepatic oxidative stress are present in both fatty liver and NASH, but NASH alone is associated with mitochondrial structural defects32. In 2007, Kotronen evaluated liver fat content by proton magnetic resonance spectroscopy in 271 nondiabetic subjects and found that liver fat content is signifcantly (4-fold higher) increased in subjects with the metabolic syndrome as compared with those without the syndrome, independently of age, gender and body mass index33. In 2010, Vanni was one of the frst to describe the bidirectional relationship linking NAFLD with metabolic syndrome and indicated that hyperinsulinemia is probably the consequence rather than cause of NAFLD34. Then Zhang in 2015 described further the bidirectional relationship; his study fndings suggested a reciprocal causality between NAFLD and metabolic syndrome35. 1.3.3 Apoptosis, Lipid and Bile Acid Metabolism In 2003, Gregory Gore and colleagues concluded that hepatocyte apoptosis is signifcantly increased in patients with nonalcoholic steatohepatitis and that it correlates with disease severity36. This study demonstrated that saturated fatty acids could induce apoptosis and were increased in patients with NASH. Another study in 2012 showed inappropriate increase in hepatic synthesis and dysregulation of cholesterol metabolism being associated with NAFLD especially elevated free cholesterol levels37. In 2011, Lima Cabello and colleagues described the potential role of liver X receptero (LXRα), a nuclear receptor that binds ligands such as cholesterol derivatives and polyunsaturated fatty acids, in the pathogenesis of NAFLD. This study showed increased hepatic expression levels of LXRα and related lipogenic and infammatory mediators compared to farnesoid X receptor (FXR), another nuclear receptor that binds bile acids (Bas) as ligands with pleotrpic effects in the liver38. 3

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

Insulin resistance, the principal risk factor for NAFLD, was found to be associated with a shift in the circulating BA profle toward a more trihydroxylic one, which has weaker FXR agonist effects compared to more hydrophobic bile acids39. These studies have provided the basis for the use of FXR agonists for the treatment of nonalcoholic steatohepatitis. 1.3.4 Infammatory Response In 1988, the 2-hit hypothesis was proposed by Day and colleagues, and it provided the basis for the current understanding of NAFLD pathophysiology, i.e., steatosis as the frst hit and the infammatory response leading to cell death as the second hit 40. In the early 2000s, multiple studies investigated the role of mitochondrial abnormalities in NAFLD. In 2000, Robertson indicated that CYPs 2E1 and 4A, which are the microsomal oxidases involved with fatty acid oxidation, could generate the “second hit” of cellular injury by creating oxidative stress when antioxidant reserve is depleted41. In 2001 a study by Sanyal et al. showed that peripheral insulin resistance, increased fatty acid beta-oxidation and hepatic oxidative stress are present in both fatty liver and NASH, but NASH alone is associated with mitochondrial structural defects32. In 2005, Zobair M. Younossi published a study describing the molecular pathogenesis and the genomic/ proteomic analysis of NAFLD. The study was done on liver biopsy specimens from 98 bariatric surgery patients who were classifed as normal, steatosis alone, steatosis with nonspecifc infammation and NASH. The genomic/ proteomic analysis of these specimens suggested differential expression of several genes and protein peaks in patients within the spectrum of NAFLD42. In 2004 and 2005, two studies reported lower plasma adiponectin concentration in NAFLD. Adiponectin is an adipokine with anti-infammatory and anti-steatotic properties. In 2006, Giovanni Targher conducted a cross-sectional study investigating the role of adiponectin in NAFLD pathogenesis. The study enrolled 60 NAFLD patients and 60 age-, sex- and body mass index (BMI)-matched healthy controls. NAFLD patients had markedly lower plasma adiponectin concentrations than control subjects. Hypoadiponectinemia was also associated with the severity of the histologic features of NASH43–45. Development of NASH involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Gyorgy Baffy was one of the frst to describe the role of Kupffer cells in the progression of NAFLD. His paper in 2009 indicated that toll-like receptors, in particular TLR4, represent a major conduit for danger recognition linked to Kupffer cell activation. A toll-like receptor is a pattern recognition receptor that recognizes bacteriaderived cytosine phosphate guanine (CpG)-containing DNA and DNA from damaged cells to activate innate immunity46. A study done by Miura K. Kodama and colleagues in 2010 showed that in a mouse model of NASH, TLR9 signaling induces production of interleukin IL-1beta by Kupffer cells, leading to steatosis, infammation and fbrosis47. Another study by Miura and colleagues concluded in 2012 that the CC-chemokine receptor (CCR)2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes48. 4

1.3.5 Genetics of NAFLD The heritability of NAFLD is supported by a body of evidence derived from epidemiological studies and genetic studies. Epidemiological studies demonstrate large interethnic variability in an individual’s susceptibility to NAFLD and NASH. A retrospective study by Stephen Caldwell in 2002 showed that the prevalence of cirrhosis attributed to NAFLD differs between different ethnic groups49. Then another study by Jeffrey Browning in 2004 showed that the ethnic differences in the frequency of hepatic steatosis mirror those observed previously for NAFLD-related cirrhosis (Hispanics > Whites > Blacks)50. A meta-analysis by Zobair M. Younossi in 2016 showed that the global prevalence of NAFLD is 25.24% (95% CI: 22.10–28.65). The highest prevalence of NAFLD is reported from the Middle East (31.79% [95% CI, 13.48–58.23]) and South America (30.45% [95% CI, 22.74–39.440]), whereas the lowest prevalence rate is reported from Africa (13.48% [5.69–28.69])3. A study done by Schwimmer and colleagues in 2009 showed that fatty liver was signifcantly more common in siblings (59%) and parents (78%) of children with NAFLD. The association was independent of adiposity and led to estimates that between 38 and 100% of hepatic fat content and NAFLD variability are due to inherited factors51. A cross-sectional analysis by Rohit Loomba in 2015 including 60 pairs of twins showed that the presence of hepatic steatosis and the level of hepatic fbrosis, as measured using magnetic resonance imaging, are correlated between monozygotic twins but not between dizygotic twins52. Multiple genome-wide association studies (GWAS) were done during the last 20 years that have expanded the understanding of genetic background contributing to variation in NAFLD susceptibility, progression and outcomes. The frst genome-wide association study of GWAS in hepatology aimed at investigating the genetic basis of susceptibility to NAFLD dates back to 2008. A study by Stephano Romeo and colleagues showed an allele in the patatin-like phospholipase domain-containing protein 3 (PNPLA3 — rs738409[G], encoding I148M) strongly associated with increased hepatic fat levels and with hepatic infammation. The allele was most common in Hispanics, the group most susceptible to NAFLD; hepatic fat content was more than 2-fold higher in PNPLA3 rs738409[G] homozygotes than in noncarriers. Resequencing revealed another allele of PNPLA3 (rs6006460[T], encoding S453I) that was associated with lower hepatic fat content in African Americans, the group at lowest risk of NAFLD53. Since 2008, other variants in different genes were found to be associated with the development of NAFLD (Table 1.1). All these genes encode proteins involved in the regulation of hepatic lipid metabolism. These include patatin-like phospholipase domain-containing protein 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), glucokinase regulator (GCKR) and membranebound O acyltransferase 7 (MBOAT7)54. Clinical implementation of genetics in NAFLD has a great potential. The rapidly growing knowledge of the genetic background will help accelerate the development of pharmacotherapies and identify targets that are likely to translate to clinical beneft. NAFLD genetics have also been incorporated into diagnostic and prognostic models and polygenic scores54,55.

1 HISTORICAL PERSPECTIVES AND CLINICAL PRESENTATION

Table 1.1: Genetic Variants That Are Associated with the Development and Progression on NAFLD Gene PNPLA3 TM6SF2 GCKR MBOAT7

Variant rs738409 C>G rs58542926 C>T rs1260326 C>T rs641738 C>T

Function Lipid droplets remodeling VLDL secretion Regulation of de novo lipogenesis Fatty acid remodeling

1.4 HISTORICAL PERSPECTIVES OF DIAGNOSTIC TESTING Diagnosis of steatosis can be done accurately using noninvasive tests56,57. However, diagnosis of steatohepatitis still requires liver biopsy. Liver biopsy has also traditionally been considered the reference method for evaluation of hepatic fbrosis. In 1999, the frst grading and staging system for histologic diagnosis of NASH was developed by Brunt and colleagues. It included 10 histological variables of disease activity based on an observed progressive increase in steatosis, ballooning and lobular infammation and a staging score for fbrosis58. In 2005, the Pathology Committee of the NASH Clinical Research Network designed and validated a histological feature scoring system that addresses the full spectrum of lesions of NAFLD and proposed the NAFLD activity score (NAS) to diagnose NASH especially for clinical trials (Table 1.2). The scoring system comprised 14 histological features, 4 of which were evaluated semiquantitatively: steatosis (0–3), lobular infammation (0–2), hepatocellular ballooning (0–2) and fbrosis (0–4). NAS of > or = 5 correlated with a diagnosis of NASH, and biopsies with scores of less than 3 were diagnosed as “not NASH”59. In 2012, another scoring system for histological severity of NAFLD was developed, called the “SAF score,” which stands for steatosis, activity and fbrosis. The SAF score used the same components as NAS, including steatosis (0–3), activity grade (A, 0–4) calculated by the unweighted addition of hepatocyte ballooning (0–2) and lobular infammation (0–2), and the fbrosis stage (0–4). However, it is reported with subscripts for each component, i.e., S (0–3) A (0–4) F (0–4), whereas the NAS is reported as a single numeric value, the unweighted sum of steatosis + lobular infammation + ballooning. Diagnosis of NASH was based on fatty liver inhibition of progression (FLIP) algorithm (Figure 1.1). The FLIP algorithm based on the SAF score has been shown to decrease interobserver variations among pathologists compared to NAS60. In 2012, Alkhouri and colleagues introduced a histological grading score for pediatric NAFLD to account for the fact that portal-based injury is a key feature of the disease in children. The score was based on steatosis, lobular infammation, ballooning and portal infammation. These histological feature were scored: steatosis (0–3), lobular infammation (0–3), ballooning (0–2) and portal infammation (0–2). This score was called the Pediatric NAFLD Histological Score (PNHS)61. Although liver biopsy is the most reliable approach for identifying the presence of steatohepatitis (SH) and fbrosis in patients with NAFLD, the biopsy specimen size has to be long enough and has to be interpreted by experts to provide reliable information62. It has also been shown that

Phenotype ↑NAFLD, NASH, fbrosis, HCC ↑NAFLD, NASH, fbrosis ↑NAFLD, NASH, fbrosis ↑NAFLD, NASH, fbrosis, HCC

Table 1.2: NAFLD Activity Score (NAS) and Its Components NAFLD Activity Score (NAS) Histologic Feature Steatosis

Hepatocyte ballooning Lobular infammation

Fibrosis stage

Score 0 66% 0 None 1 Few 2 Many 0 None 1 1–2 foci per 20× feld 2 2–4 foci per 20× feld 3 >4 foci per 20× feld 0 No fbrosis 1a Zone 3 mild perisinusoidal fbrosis 1b Zone 3 moderate perisinusoidal fbrosis 1c portal or periportal fbrosis 2 Zone 3 and portal/periportal fbrosis 3 Bridging fbrosis 4 Cirrhosis

there is variability among hepatopathologists’ readings for NASH histologic features and overall diagnosis63. Besides technical problems, liver biopsy remains a costly and an invasive procedure. These limitations led to the evolution of noninvasive testing for assessment of liver disease severity and fbrosis. Noninvasive tests can be classifed into (1) blood-based tests and (2) imaging methods assessing physical properties of the liver tissue, including stiffness attenuation and viscosity. Numerous recent publications have reported on the accuracy of existing and novel NITs to assess liver diseases. These will be discussed in detail in another chapter. 1.5 HISTORICAL PERSPECTIVES OF THERAPEUTICS As our understanding of NAFLD pathophysiology is advancing rapidly, the development of therapeutics that can affect a variety of new targets has been progressing. The treatment of NAFLD can be divided into lifestyle modifcations to induce weight loss and pharmacological treatments. In a meta-analysis of eight randomized, controlled trials (RCTs), published in 2012, 5% weight loss led to improvement in steatosis, whereas 7% weight loss was associated with an improved NAS and resolution of NASH64. Another study published in 2015 of 293 patients 5

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

Steatosis

Ballooning

0

1, 2, 3

1

2

Figure 1.1

Diagnosis

0

NAFLD

1

NAFLD

2

NAFLD

0

NAFLD

1

NASH

2

NASH

0

NAFLD

1

NASH

2

NASH

Fatty liver inhibition of progression (FLIP) algorithm based on the steatosis/activity/fbrosis (SAF) score

with histologically proven NASH showed that the highest rates of NAS reduction, NASH resolution and fbrosis regression occurred in patients with weight losses ≥10%65. However, only 50% of patients achieved 7% or more weight loss at 12 months and only 10% of patients lost ≥10%. Given that patients with NAFLD without SH or any fbrosis have an excellent prognosis from a liver standpoint, pharmacological treatments aimed primarily at improving liver disease should generally be limited to those with biopsy-proven NASH and fbrosis1. The antioxidant vitamin E was one of the frst drugs to be tested for NASH. A pilot study in 2000 showed that vitamin E administration normalized serum aminotransferases and alkaline phosphatase levels in children. Another pilot study in 2004 studied the combination of antioxidant (vitamin E) and an insulin sensitizer (pioglitazone) and showed that vitamin E alone produced a signifcant decrease in steatosis, whereas combination treatment produced improvement in NASH histology including decrease in steatosis cytological ballooning and pericellular fbrosis66,67. The PEVINS trial in 2010 included 247 individuals with NASH and without diabetes who were randomly assigned to receive pioglitazone (30 mg daily), vitamin E (800 IU daily) or placebo. Vitamin E was associated with a higher rate of improvement in NAS by 2 points or more than placebo, whereas the rate of improvement with pioglitazone was not signifcant. Individuals who received pioglitazone gained more weight than those 6

Lobular Inflammation

who received vitamin E or placebo. Otherwise, other the side effects were similar68. The widespread use of vitamin E was limited due to concerns about the association with hemorrhagic stroke risk in observational studies69. Another concern was the association with prostate cancer observed from continued followup of individuals in the SELECT trial, which showed that the absolute increase in risk of prostate cancer per 1000 person-years was 1.6 for vitamin E translating into a 17% incrased risk70. Weight gain, which is the most common side effect of pioglitazone, and the possible association of pioglitazone with bladder cancer and bone loss have limited the use of pioglitazone by providers71,72. Omega-3 fatty acids have been investigated to treat NAFLD. In a literature review in 2010 by Masterton and colleagues, animal studies demonstrated that omega-3 fatty acids reduced hepatic steatosis, improved insulin sensitivity and reduced markers of infammation; however, clinical trials in human subjects were limited by small sample size and methodological faws73. Two other studies failed to show convincing therapeutic beneft for omega-3 fatty acids74,75. The American Association for the Study of Liver Disease (AASLD) NAFLD guidelines recommend against using Omega-3 fatty acids as a specifc treatment of NAFLD or NASH, but they may be considered to treat hypertriglyceridemia in patients with NAFLD1. Currently, several emerging therapies have progressed to phase II and beyond, and they will be discussed in detail in another chapter.

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17. Kim, D. et al. Metabolic dysfunction-associated fatty liver disease is associated with increased all-cause mortality in the United States. Journal of Hepatology 75, 1284–1291 (2021). 18. Younossi, Z. M. et al. From NAFLD to MAFLD: Implications of a premature change in terminology. Hepatology (Baltimore, Md.) 73, 1194–1198 (2021). 19. Enzi, G., Busetto, L., Inelmen, E. M., Coin, A. & Sergi, G. Historical perspective: Visceral obesity and related comorbidity in Joannes Baptista Morgagni’s “De sedibus et causis morborum per anatomen indagata”. International Journal of Obesity and Related Metabolic Disorders : Journal of the International Association for the Study of Obesity 27, 534–535 (2003). 20. Haslam, D. Diabesity-a historical perspective: Part II Article points. 21. Avogaro, P., Crepaldi, G., Enzi, G. & Tiengo, A. Associazione di iperlipemia, diabete mellito e obesita’ di mediogrado. Acta Diabetologica Latina 4, 572–590 (1967). 22. Feldman, R., Sender, A. J. & Siegelaub, A. B. Difference in diabetic and nondiabetic fat distribution patterns by skinfold measurements. Diabetes 18, 478–486 (1969). 23. Kissebah, A. H. et al. Relation of body fat distribution to metabolic complications of obesity. The Journal of Clinical Endocrinology and Metabolism 54, 254–260 (1982). 24. Reaven, G. M. Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37, 1595–1607 (1988). 25. Zelman, S. The liver in obesity. A.M.A. Archives of Internal Medicine 90, 141–156 (1952). 26. Beringer, A. & Thaler, H. Relationships between diabetes mellitus and fatty liver. Deutsche medizinische Wochenschrift (1946) 95, 836–838 (1970). 27. Itoh, S., Tsukada, Y., Motomura, Y. & Ichinoe, A. Five patients with nonalcoholic diabetic cirrhosis. Acta hepato-gastroenterologica 26, 90–97 (1979). 28. Powell, E. E. et al. The natural history of nonalcoholic steatohepatitis: A follow-up study of forty-two patients for up to 21 years. Hepatology (Baltimore, Md.) 11, 74–80 (1990). 29. Powell, E. E., Searle, J. & Mortimer, R. Steatohepatitis associated with limb lipodystrophy. Gastroenterology 97, 1022–1024 (1989). 30. Bacon, B. R., Farahvash, M. J., Janney, C. G. & Neuschwander-Tetri, B. A. Nonalcoholic steatohepatitis: An expanded clinical entity. Gastroenterology 107, 1103–1109 (1994). 31. Marchesini, G. et al. Association of nonalcoholic fatty liver disease with insulin resistance. The American Journal of Medicine 107, 450–455 (1999). 32. Sanyal, A. J. et al. Nonalcoholic steatohepatitis: Association of insulin resistance and mitochondrial abnormalities. Gastroenterology 120, 1183–1192 (2001). 33. Kotronen, A., Westerbacka, J., Bergholm, R., Pietiläinen, K. H. & Yki-Järvinen, H. Liver fat in the metabolic syndrome. The Journal of Clinical Endocrinology and Metabolism 92, 3490–3497 (2007). 34. Vanni, E. et al. From the metabolic syndrome to NAFLD or vice versa? Digestive and Liver Disease: Offcial Journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver 42, 320–330 (2010). 35. Zhang, Y. et al. Identification of reciprocal causality between non-alcoholic fatty liver disease and metabolic syndrome by a simplified Bayesian network in a Chinese population. BMJ Open 5, e008204 (2015). 7

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36. Feldstein, A. E. et al. Hepatocyte apoptosis and Fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 125, 437–443 (2003). 37. Min, H. K. et al. Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease. Cell Metabolism 15, 665–674 (2012). 38. Lima-Cabello, E. et al. Enhanced expression of proinfammatory mediators and liver X-receptor-regulated lipogenic genes in non-alcoholic fatty liver disease and hepatitis C. Clinical Science (London, England: 1979) 120, 239–250 (2011). 39. Haeusler, R. A., Astiarraga, B., Camastra, S., Accili, D. & Ferrannini, E. Human insulin resistance is associated with increased plasma levels of 12α-hydroxylated bile acids. Diabetes 62, 4184–4191 (2013). 40. Day, C. P. & James, O. F. Steatohepatitis: A tale of two “hits”? Gastroenterology 114, 842–845 (1998). 41. Robertson, G., Leclercq, I. & Farrell, G. C. Nonalcoholic steatosis and steatohepatitis. II. Cytochrome P-450 enzymes and oxidative stress. American Journal of Physiology. Gastrointestinal and Liver Physiology 281, G1135–G1139 (2001). 42. Younossi, Z. M. et al. A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease. Hepatology (Baltimore, Md.) 42, 665–674 (2005). 43. Hui, J. M. et al. Beyond insulin resistance in NASH: TNF-alpha or adiponectin? Hepatology (Baltimore, Md.) 40, 46–54 (2004). 44. Pagano, C. et al. Plasma adiponectin is decreased in nonalcoholic fatty liver disease. European Journal of Endocrinology 152, 113–118 (2005). 45. Targher, G. et al. Associations between plasma adiponectin concentrations and liver histology in patients with nonalcoholic fatty liver disease. Clinical Endocrinology 64, 679–683 (2006). 46. Baffy, G. Kupffer cells in non-alcoholic fatty liver disease: The emerging view. Journal of Hepatology 51, 212–223 (2009). 47. Miura, K. et al. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 139, 323–334.e7 (2010). 48. Miura, K., Yang, L., van Rooijen, N., Ohnishi, H. & Seki, E. Hepatic recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2. American Journal of Physiology. Gastrointestinal and Liver Physiology 302, G1310–G1321 (2012). 49. Caldwell, S. H., Harris, D. M., Patrie, J. T. & Hespenheide, E. E. Is NASH underdiagnosed among African Americans? The American Journal of Gastroenterology 97, 1496–1500 (2002). 50. Browning, J. D. et al. Prevalence of hepatic steatosis in an urban population in the United States: Impact of ethnicity. Hepatology (Baltimore, Md.) 40, 1387–1395 (2004). 51. Schwimmer, J. B. et al. Heritability of nonalcoholic fatty liver disease. Gastroenterology 136, 1585–1592 (2009). 52. Loomba, R. et al. Heritability of hepatic fbrosis and steatosis based on a prospective twin study. Gastroenterology 149, 1784–1793 (2015). 53. Romeo, S. et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nature Genetics 40, 1461–1465 (2008). 54. Eslam, M., Valenti, L. & Romeo, S. Genetics and epigenetics of NAFLD and NASH: Clinical impact. Journal of Hepatology 68, 268–279 (2018). 8

55. Eslam, M. et al. FibroGENE: A gene-based model for staging liver fbrosis. Journal of Hepatology 64, 390–398 (2016). 56. Idilman, I. S. et al. Hepatic steatosis: Quantifcation by proton density fat fraction with MR imaging versus liver biopsy. Radiology 267, 767–775 (2013). 57. Siddiqui, M. S. et al. Vibration-controlled transient elastography to assess fbrosis and steatosis in patients with nonalcoholic fatty liver disease. Clinical Gastroenterology and Hepatology 17, 156–163.e2 (2019). 58. Brunt, E. M., Janney, C. G., di Bisceglie, A. M., Neuschwander-Tetri, B. A. & Bacon, B. R. Nonalcoholic steatohepatitis: A proposal for grading and staging the histological lesions. The American Journal of Gastroenterology 94, 2467–2474 (1999). 59. Kleiner, D. E. et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology (Baltimore, Md.) 41, 1313–1321 (2005). 60. Bedossa, P. & FLIP Pathology Consortium. Utility and appropriateness of the fatty liver inhibition of progression (FLIP) algorithm and steatosis, activity, and fbrosis (SAF) score in the evaluation of biopsies of nonalcoholic fatty liver disease. Hepatology (Baltimore, Md.) 60, 565–575 (2014). 61. Alkhouri, N. et al. Development and validation of a new histological score for pediatric non-alcoholic fatty liver disease. Journal of Hepatology 57, 1312–1318 (2012). 62. Bedossa, P., Dargère, D. & Paradis, V. Sampling variability of liver fbrosis in chronic hepatitis C. Hepatology (Baltimore, Md.) 38, 1449–1457 (2003). 63. Davison, B. A. et al. Suboptimal reliability of liver biopsy evaluation has implications for randomized clinical trials. Journal of Hepatology 73, 1322–1332 (2020). 64. Musso, G., Cassader, M., Rosina, F. & Gambino, R. Impact of current treatments on liver disease, glucose metabolism and cardiovascular risk in non-alcoholic fatty liver disease (NAFLD): A systematic review and meta-analysis of randomised trials. Diabetologia 55, 885–904 (2012). 65. Vilar-Gomez, E. et al. Weight loss through lifestyle modifcation signifcantly reduces features of nonalcoholic steatohepatitis. Gastroenterology 149, 367–378.e5; quiz e14–15 (2015). 66. Sanyal, A. J. et al. A pilot study of vitamin E versus vitamin E and pioglitazone for the treatment of nonalcoholic steatohepatitis. Clinical Gastroenterology and Hepatology: The Offcial Clinical Practice Journal of the American Gastroenterological Association 2, 1107–1115 (2004). 67. Lavine, J. E. Vitamin E treatment of nonalcoholic steatohepatitis in children: A pilot study. The Journal of Pediatrics 136, 734–738 (2000). 68. Sanyal, A. J. et al. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. The New England Journal of Medicine 362, 1675–1685 (2010). 69. Keli, S. O., Hertog, M. G., Feskens, E. J. & Kromhout, D. Dietary favonoids, antioxidant vitamins, and incidence of stroke: The Zutphen study. Archives of Internal Medicine 156, 637–642 (1996). 70. Klein, E. A. et al. Vitamin E and the risk of prostate cancer: The Selenium and Vitamin E Cancer Prevention Trial (SELECT). JAMA 306, 1549–1556 (2011). 71. Tang, H. et al. Pioglitazone and bladder cancer risk: A systematic review and meta-analysis. Cancer Medicine 7, 1070–1080 (2018).

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72. Yau, H., Rivera, K., Lomonaco, R. & Cusi, K. The future of thiazolidinedione therapy in the management of type 2 diabetes mellitus. Current Diabetes Reports 13, 329–341 (2013). 73. Masterton, G. S., Plevris, J. N. & Hayes, P. C. Review article: Omega-3 fatty acids—a promising novel therapy for non-alcoholic fatty liver disease. Alimentary Pharmacology & Therapeutics 31, 679–692 (2010).

74. Sanyal, A. J. et al. No signifcant effects of ethyleicosapentanoic acid on histologic features of nonalcoholic steatohepatitis in a phase 2 trial. Gastroenterology 147, 377–384.e1 (2014). 75. Scorletti, E. et al. Effects of purifed eicosapentaenoic and docosahexaenoic acids in nonalcoholic fatty liver disease: Results from the Welcome* study. Hepatology (Baltimore, Md.) 60, 1211–1221 (2014).

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2 Epidemiology and Natural History of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis Linda Henry and Zobair M. Younossi

CONTENTS 2.1 General Prevalence of NAFLD and NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2 Prevalence of NAFLD According to Age, Gender and Ethnicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.3 Prevalence of NAFLD in Subpopulations (Diabetes Mellitus, Obese, Metabolic Syndrome, Lean) (Figure 2.3) . . . . . 12 2.4 Prevalence of Fibrosis in NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.5 Incidence of NAFLD, NASH and Related Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.6 Natural History of NAFLD and NASH (Figure 2.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.7 Risk Factors for NAFLD and NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 2.8 NAFLD Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.1 GENERAL PREVALENCE OF NAFLD AND NASH Nonalcoholic fatty liver disease (NAFLD) is described as a spectrum of fatty liver disease that is associated with components of metabolic syndrome including obesity, insulin resistance, type 2 diabetes mellitus (T2DM), hypertension, and dyslipidemia. [1,2] NAFLD is diagnosed when the presence of hepatic steatosis affects at least 5% of liver hepatocytes in the absence of other causes of liver disease and excess alcohol consumption. [3] The histologic spectrum includes simple hepatic steatosis without other histologic changes (NAFL) as well as nonalcoholic steatohepatitis (NASH), which includes hepatic steatosis in addition to ongoing liver cell injury that can potentially lead to fbrosis, cirrhosis and hepatocellular carcinoma (HCC). [1–3] Presence of insulin resistance, type 2 diabetes mellitus (T2DM) and visceral obesity not only increases the risk of hepatic steatosis but also is associated with the severity of liver disease, including development of NASH and increasing the stage of hepatic fbrosis. In this context, the relationship of T2DM and NAFLD is bidirectional. For example, one recent meta-analysis determined that NAFLD is associated with an almost 2.2-fold increased risk of incident diabetes and that this risk rises as NAFLD becomes severe. [4] In addition, diseases that are associated with T2DM are also more common in patients with NAFLD. In this context, several other studies recently reported that NAFLD is associated with a 1.5 times increased risk for the development of chronic kidney disease (CKD) ≥ stage 3 and an almost 1.5- to 2.0-fold increased risk for the development of extrahepatic cancers (GI cancers, breast cancer and gynecological cancers). [5,6] Given this association and the dramatic increase in the prevalence of obesity and T2DM over the past several decades as well as improvements in care of viral hepatitis, NAFLD is now poised to become the most common chronic liver disease in many regions of the world. [7,8] Currently, it is estimated that 25–30% of the global general adult population has NAFLD. [9] Additionally, it is estimated that the prevalence of NASH in the general population is approximately 5–6%. [1,10,11] It is important to note that these rates are signifcantly higher among some patient populations. In fact, among those undergoing weight reduction surgeries, the prevalence of NAFLD can be over 80%, while among those with type 2 diabetes mellitus, the prevalence of NAFLD is over 55% with over a third (37.3%) of diabetics estimated to 10

have NASH and 17% estimated to have advanced fbrosis. [12,13] As better and more accurate noninvasive tests for NASH become available, our understanding of the true prevalence and incidence of this disease state will be forthcoming. In addition to negative impact on clinical outcomes, NAFLD has been shown to have a negative impact of patient reported outcomes (PROs), including health-related quality of life, the ability to be physically active, the ability to be present at work, increased fatigue and depression. [14–22] Furthermore, the economic burden of NAFLD has been reported to be quite substantial especially among those with NASH and fbrosis. [23–29] However, there are geographic variations in the prevalence of NAFLD (Figure 2.1).[30] The overall prevalence of NAFLD in Asia is estimated to be 29.6%. [31] The prevalence in China is reported to be approximately 29.2%, whereas Japan also now reports a prevalence of between 25 and 35%, an over 2-fold increase from the 1990s. [32] The overall NASH prevalence is estimated to be between 1.9 and 2.7% among the general population, but this rate may be very underreported given the diffculties in the diagnosis of NASH, i.e., liver biopsy. [33] The rates of NASH are much higher in those with NAFLD. For example, one study from China reported that the prevalence of NASH in patients with biopsy-proven NAFLD was 58.9%, while others reported NASH prevalence rates as high as 97.4 and 93.8% in Singaporean and Japanese NAFLD cohorts, respectively. [34] In addition to the East Asian countries, the prevalence of NAFLD and NASH has also been reported from South Asia. [10,35–40] India has a reported prevalence rate that ranges from 8.7 to 32.6%, depending on whether patients were from rural or urban areas, respectively. [37] In urban Sri Lanka, the prevalence of NAFLD was reported to be 32.6%. [38] The overall prevalence in the Middle East has been reported to range from 20 to 30%. [41,42] The prevalence of NAFLD in Kuwait, Saudi Arabia, South of Iran and North of Iran was reported as 33.3, 16.6, 21.5 and 43.8%, respectively. [41,42] Israel reports a NAFLD prevalence of 30%. [43] A recent study completed in Nigeria found that the overall prevalence of NAFLD was 8.7%, while for Sudan the reported rate was 20%. [44,45] These results are in line with what has been reported in a prior study in which the NAFLD prevalence for Africa was estimated at 13–14%. [9] DOI: 10.1201/9781003386698-3

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Figure 2.1

Geographic variations in the prevalence of NAFLD

In Europe, the prevalence of NAFLD is around 24%. [9] There is a wide range of prevalence rates reported from different European countries. Greece reports NAFLD prevalence of 41%, while Spain reported a prevalence of NAFLD of 33% in men and 20% in women. [46,47] In Australia, the prevalence of NAFLD has been reported to be 20–30% and is the most common liver disease in Australia. [48] In New Zealand, the prevalence of NAFLD was reported to be 28% in 2015. [49] Cuba reports a prevalence of approximately 17%. [50] Current estimates suggest the prevalence of NASH is approximately 5–6% among the general population. [9,40] Although most of the data regarding the epidemiology of NAFLD in North America come from studies carried out in the United States (US), the estimated NAFLD prevalence rate is 24.13%. [9,10] The prevalence of NAFLD in the United States ranges between 18.8 and 40%. [11,30,50–53] On the other hand, based on limited ultrasound data, the prevalence of NAFLD in Mexico ranges between 14.4 and 62.9%. [54–56] Similarly, the prevalence of NAFLD (diagnosed by imaging) in Canada is estimated to be around 24.7%. [13,57] Recent data from Central and South America are lacking, but one study suggested that, based on the rates of obesity, the prevalence of NAFLD was estimated as 26% in Mexico; between 15 and 20% in Central America; 28% in Belize and Barbados; 24% in Venezuela and Chile; 20% in Uruguay; 18% in Guyana, Paraguay and Ecuador; and ≤16% in the other countries. [58] As noted, the true prevalence of NASH by geographical region is not known from population-based studies; however, to fll the gap, investigators have undertaken the use of modeling to determine the NASH prevalence. One such study conducted in 2016 demonstrated that the prevalence of NASH within the general population (to include children) ranged from 2.4% for China to 5.3% for the US, while among those with NAFLD, the prevalence

of NASH ranged from 13.4% for China to 20.3% for the US, but rates were forecasted to be 7.6% in the general US population, followed by 6.3% for Italy and 3.4% for China. [59] In 2017, using the same modeling techniques for Saudi Arabia and the United Arab Emirates (UAE), the NASH prevalence was estimated to be 4.2 and 4.1%. [60] Similar studies have been done for Hong Kong, Singapore, South Korea, Taiwan, Australia, Canada and Switzerland, with all studies noting that if the rates of obesity and type 2 diabetes mellitus continue to increase, the prevalence of NAFLD and NASH will increase as well, and in some cases the incidence of NASH will outpace that of NAFLD. [61–64] The investigators also reported that by 2030, NASH in the general population is expected to be greater than 6% an increase between 87 and 96%. [65] 2.2 PREVALENCE OF NAFLD ACCORDING TO AGE, GENDER AND ETHNICITY The consensus is that the prevalence of NAFLD increases with age. The peak prevalence of NAFLD for males is between ages 50 and 60 years (29.3%) [64], while for females the peak time is noted for those over the age of 65 (25.4%). [66,67] Based on NHANES III data, the prevalence rates for males by age have been cited as 16.1% in those aged 30–40 years old and 22.3% in those aged 41–50 years old, and 27.6% in those over 60 years old. [68] For women, the prevalence of NAFLD was 12.5% in those aged 30–40 years old and 16.1% in those aged 41–50 years old and 21.6% for those 51–60 years. [68] Assessment of disease burden according to gender, researchers found that women 50 years and older were 17% more likely to develop NASH and 56% more likely to develop advanced fbrosis compared to males of similar ages. [69] Ethnicity can also impact the prevalence and severity of NAFLD. In the United States, one study found the overall NAFLD prevalence was 32.6% and was highest among 11

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Mexican Americans at 48.4% and lowest in non-Hispanic Blacks (18.0%) and Asians (18.1%), while non-Hispanic whites had a prevalence of 33% and Hispanics outside of Mexican Americans had a prevalence of 32%. However, a disturbing fnding was that, despite having the lowest prevalence of NAFLD, non-Hispanic Blacks (28.5%) had the highest prevalence of advanced fbrosis, while nonHispanic Asians had the lowest (2.7%).[70] Another study that explored the NAFLD prevalence among different subgroups of minorities (Japanese Americans, Whites, Latinos, African Americans and Native Hawaiians) in the United States found that NAFLD accounted for over 50% of the cases of chronic liver disease (CLD) and that 63.6% of the Japanese Americans with CLD had NAFLD, followed by 57.8% of Native Hawaiians, 45.6% of Latinos, 40.7% of whites and 39.2% of African Americans. [71] NALFD was also the most common cause of cirrhosis, accounting for over 32% of cases. When stratifed by race/ ethnicity, NAFLD accounted for 32.3% of cirrhosis cases in Japanese Americans, 31.5% in Native Hawaiians, and 31.9% in Latinos. Interestingly, in a study using NHANES data in which NAFLD was diagnosed by FibroScan® and controlled attenuation parameter, Mexican Americans had the highest prevalence of moderate to severe steatosis, while African Americans had the lowest, but when the investigators looked by gender, it was male Mexican Americans that had a higher likelihood of having moderate to severe steatosis. [72] In a meta-analysis, similar results were found where Hispanics had the higher prevalence of both NAFLD and NASH, but the proportion of patients with signifcant fbrosis did not signifcantly differ among racial or ethnic groups. [73] The role of ethnicity was also explored outside the United States. In a recent meta-analysis from China, the NAFLD prevalence of 29.2% by ethnicity found the Hui had the highest NAFLD prevalence of 53.8%, followed by the Uygur at 46.6%, then Taiwan at 39.9% and northwest China at 39.9%. [33] (See Figure 2.2.)

Figure 2.2 12

NAFLD prevalence by ethnicity

Finally, among the younger age groups, a recent metaanalysis of 14 studies of the prevalence of NAFLD among children and adolescents reported an overall prevalence of 7.4% regardless of the diagnostic method used and 8.8% when ultrasound was used. The investigators also reported the prevalence of NAFLD by global areas and found a NAFLD prevalence of 8.5% for North America, 7.0% for Asia and 1.7% for Europe. Like adults, the prevalence of NAFLD was higher among those with obesity at 52.5%. Their trend analysis indicated that NAFLD prevalence is increasing at 0.26% per year, and it forecasts that by 2040, the NAFLD prevalence among the youth will be almost 31%. [74] 2.3 PREVALENCE OF NAFLD IN SUBPOPULATIONS (DIABETES MELLITUS, OBESE, METABOLIC SYNDROME, LEAN) (FIGURE 2.3) Although the prevalence of NAFLD and NASH is quite high in the general population, it is even higher in specifc cohorts, such as those with T2DM and the severely obese. In a systematic review of studies, the overall global prevalence of NAFLD among patients with T2DM was 61.1%. [13] The prevalence of NASH among those with diabetes and NAFLD was found to be as high 64.0%. Furthermore, the prevalence of advanced fbrosis (fbrosis, ≥ F3) was also found to be high at 10.4%. [13] In a study from China, the prevalence of NAFLD among those with T2DM was 51.8% as compared to 30.8% in nondiabetics. [75] NAFLD has also been reported for those with type 1 diabetes. A metaanalysis of studies conducted for those with type 1 diabetes found an overall pooled NAFLD prevalence of 19.3% in which the prevalence was 22.0% in adults only and 7.9% in children/adolescents. [76] In a study conducted among morbidly obese patients undergoing weight reduction surgery, the prevalence of NAFLD was found to be 95%. [77] Furthermore, a study from China noted that the prevalence of NAFLD among

2 EPIDEMIOLOGY AND NATURAL HISTORY

Figure 2.3

NAFLD prevalence by subgroups

those with obesity was 66.2% compared to only 11.7% in those of normal weight and that the higher prevalence of NAFLD was found in the areas with a higher gross domestic product. [75] NAFLD has also been reported in those considered nonobese or “lean” with a reported prevalence of 11.2% in the general population and a reported prevalence among those with NAFLD of 25.3% with a range of 19.2% among the lean and 41% among the nonobese (lean and overweight). The prevalence also varied by geographical area from 25% or lower in some countries (e.g., Malaysia and Pakistan) to higher than 50% in others (e.g., Austria, Mexico and Sweden). [78] This group appears to carry a higher burden of comorbidities as well as a higher risk of mortality than those without NAFLD. [79–82] Although the lean group has less severe histologic fndings, including hepatocyte ballooning, lobular infammation, NAFLD activity score and fbrosis stage, they may be at higher risk for severe liver disease and possibly mortality suggesting that lean NAFLD is a distinct entity with metabolic, biochemical and infammatory abnormalities compared to healthy subjects. [79] In fact, a recent meta-analysis reported that, among those with lean (nonobese) NAFLD, 39% had NASH, 29% had fbrosis stage ≥2, and 3.2% had cirrhosis, along with a reported incidence for all-cause mortality at 12.1 per 1000 person-years, liver-related mortality at 4.1 per 1000 person-years, cardiovascular-related mortality at 4.0 per 1000 person-years, new-onset diabetes at 12.6 per 1000 person-years, and new-onset hypertension at 56.0 per 1000 person-years. [80] 2.4 PREVALENCE OF FIBROSIS IN NAFLD Since the fbrosis stage is the most important predictor for adverse outcomes in those with NAFLD, recognizing the prevalence rate among those with NAFLD is imperative so that timely treatment and referrals can be initiated. [83] In a study using data from NHANES (2005–2016), the prevalence of NAFLD-related advanced fbrosis doubled over a decade with a steady increase in the prevalence noted for Hispanics and non-Hispanic Whites but not for non-Hispanic Blacks. [84] Similar studies conducted in Asia reported rates of advanced fbrosis among those with NAFLD ranging between 4 up to 25%. [33,85] In a study of over 1000 residents in Cuba, cirrhosis was diagnosed

in 3.5% of the patients at the time of their NAFLD diagnosis. [86] A meta-analysis from South Korea found that, although NAFLD and NASH were quite prevalent, the prevalence of advanced fbrosis was low (18%) but that over time the progression of disease may occur, increasing the disease burden. [87] The prevalence of advanced fbrosis among those with NASH and NASH HCC has also been studied and reported to range from 21 to 50% for patients with NASH and from 44 to 80% of patients with NASH-HCC. [88,89] The major independent predictors of having advanced fbrosis found in global studies were increasing age (OR:1.11) and diabetes (OR:2.28). [90] Other investigators have also suggested that it was the presence of a higher percentage of body fat and waist circumference that were signifcantly associated with worsening of fbrosis as measured with the NAFLD fbrosis score. [90] In a study of the global burden of cirrhosis, investigators found that over a 30-year period, the number of prevalent cases for compensated cirrhosis due to NASH doubled while the number for decompensated cirrhosis due to NASH tripled. Another study assessing the burden of NAFLD/NASH-related burden using the same Global Burden of Disease database revealed that between 2007 and 2017, cirrhosis DALY (disability-adjusted life years) due to NAFLD/NASH increased by 23.4%, while liver cancer DALY due to NAFLD/NASH increased by 37.5%. [8,91] 2.5 INCIDENCE OF NAFLD, NASH AND RELATED FIBROSIS Studies on the incidence of NAFLD are sparse partly due to the length of time it takes for the disease to be identifed and progress. In one relatively long-term study, it was noted that, during 348,193.5 person-years of follow-up, 10,340 of the participants developed NAFLD, providing an incidence rate of 29.7 per 1000 person-years. In this same study, the investigators found that those who were overweight were at 2 times the risk for developing NAFLD, while those considered obese were at almost four times the odds. [93] The presence of metabolic risk factors further increased the risk of developing NAFLD by at least 2 times even among those with normal weight. [93] In a recently reported meta-analysis, the researchers recorded several incident rates for the progression 13

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

of fbrosis, NASH, HCC and mortality. They found that almost 41% of those with NASH experienced fbrosis progression with an annual rate of progression of 0.09%. HCC incidence among NAFLD patients was noted at 0.44 per 1000 person-years with a range of 0.29–0.66 per 1000 person-years. Liver-specifc mortality among those with NAFLD was reported as 0.77 per 1000 person-years with a range of 0.33–1.77 compared to 11.77 per 1000 person-years with a range of 7.10–19.53 per 1000 person-years for those with NASH. On the other hand, overall mortality among those with NAFLD was found to be 15.44 per 1000 personyears with a range of 11.72–20.34 per 1000 person-years, and for NASH it was 25.56 per 1000 person-years with a range of 6.29–103.80 per 1000 person-years. [9] The incidence of NAFLD in the nonobese population (without NAFLD at baseline) was 24.6 (95% CI 13·4–39·2) per 1000 person-years. These rates, though lower, are still worrisome given this is a population not heavily associated with the development of NAFLD but does help raise awareness about the potential prevalence of NAFLD across the general population and not just those considered to be obese. [31] 2.6 NATURAL HISTORY OF NAFLD AND NASH (FIGURE 2.4) NAFLD is a complex disease with signifcant variability between different patients in regard to disease development, progression, and regression. Our current understanding of the disease suggests that a minority of patients with NAFLD progress to liver fbrosis or cirrhosis. Nevertheless, 10–20% of patients with NASH can progress and some of these patients can be rapid progressors. [94] Adding to the complexity of this disease is that the progression of NAFLD and NASH is not linear. In this context, some patients will progress for a period of time followed by stability or regression. In fact, it is important to note that the majority of progressors are those with insulin resistance and T2DM. [95,96,97] In one prospective study, 23% of patients with simple steatosis at baseline developed NASH in the follow-up. [98] Recently the 20%

Figure 2.4 14

Natural history of NAFLD

rule was suggested in that 20% of those with NASH and F3/F4 will progress over 2.5 years. [99] Furthermore, a recent systematic review reported that 64% of those with NAFL progressed to NASH after a mean follow-up of 3.7 years (SD 2.1). [100] Although patients with NAFLD seem to have a relatively nonprogressive course, some may develop fbrosis albeit at a signifcantly slower pace than patients with NASH. [95,101,102] 2.7 RISK FACTORS FOR NAFLD AND NASH As previously noted, NAFLD is closely associated with metabolic disorders of obesity, insulin resistance, T2DM, hypertension, mixed hyperlipidemia, and hypercholesterolemia in addition to older age and male sex. Recent studies have reported that between 28 and 66% of patients with NAFLD have metabolic syndrome. [103–107] The presence of metabolic syndrome can increase the rate of liver fbrosis progression, leading to cirrhosis, HCC, and/or death. In fact, the more components of metabolic syndrome there are, the higher the risk of mortality will be. [104,105] In fact, a review of patients with NAFLD on the waiting list for liver transplant confrmed the high prevalence of metabolic comorbidities especially for obesity, T2DM and hypertension. [108] Similar rates have been reported from other parts of the world to include Middle East, Africa, China, Cuba and Spain. [109–111] NAFLD in children is also associated with metabolic comorbidities, which include type 2 diabetes, sleep disorders, osteoporosis, vitamin D defciency, hypertension and dyslipidemia. [112] However, there is evidence for familial clustering of NAFLD. In fact, as many as 27% of cases of NAFLD may be related to familial clustering, suggesting a genetic predisposition for NAFLD and NASH development and progression. [112] More research studying NAFLD in children and families is warranted. As noted previously, ethnicity also appears to play a considerable role in the development of NAFLD, which may be partially attributed to differences in genetic background. In North America, the highest prevalence

2 EPIDEMIOLOGY AND NATURAL HISTORY

of NAFLD has been observed in Hispanics, followed by non-Hispanic white individuals, while African Americans appear to have a very low prevalence rate but a much higher mortality rate if present. [113–115] Among Hispanic patients, the genetic marker from the serpin family E member 1 (PAI-1, a marker of fbrosis) is only associated with NAFLD in Hispanic patients, whereas serum levels of adiponectin are associated with NAFLD in African Americans. [113–119] Other studies have shown that fructose malabsorption (ingestion of high amounts of fructose are associated with NAFLD) is greater in African Americans than in Hispanics. [120] The increased levels of adiponectin among African Americans may be attributed to the gene patatin-like phospholipase domaincontaining protein-3 (PNPLA3). [116] This gene encodes adiponutrin, a triacylglycerol lipase important in fatty acid hydrolysis in adipocytes. A polymorphism of I148M PNPLA3 has been associated with a higher probability of developing NAFLD. [116–119] African Americans tend to have a lower genotype distribution of PNPLA3, which may partly explain why the prevalence of NAFLD is higher in Hispanics and lower in African Americans in the United States. [115] Given the rapid increase in the prevalence of NAFLD in China, studies are also investigating the genetic link. As such, recent studies from Asia have shown a number of gene polymorphisms associated with NAFLD. [32] In particular, the PNPLA3 with the rs738409 gene has been consistently shown to increase the risk of severe steatosis, NASH and liver fbrosis in adults and appears to be more common among East Asians than Caucasians. This may be the reason behind the high NAFLD prevalence in East Asia despite a lower metabolic burden. Other genetic variants signifcantly associated with NAFLD include those in NCAN, GCKR and LYPLAL1, as well as the polymorphisms C-482T and T-455C in APOC, which has also been associated with insulin resistance. [32,116–119] Gut dysbiosis has also been implicated in the development of NAFLD. Although studies continue to further our understanding of all the mechanisms involved with the effect of gut microbiota on the organs outside the intestinal system, it is now known that these effects are achieved by various bacterial metabolites, which include bile acids, short-chain fatty acids, amino acids, choline, and ethanol. These metabolites appear to cause a chronic infammatory state and a dysregulation of lipids and branched chain amino acids. Changes in the gut fora also correspond to the state of NAFLD and NASH. [121–123] A study of children found that, among the children that had NAFLD, the alpha-diversity of gut microbiota was lower compared with their healthy controls and those with NASH had the lowest alpha-diversity. [124] Studies in this area are ongoing especially with respect to what intervention may be appropriate for a healthy gut microbiome. 2.8 NAFLD MORTALITY The progressive potential for NAFLD can confer increase mortality. Although liver-related mortality is higher among patients with NASH, the most common cause of death among patients with NAFLD is cardiovascular mortality. In this context, the overall mortality rate for NAFLD is estimated to be 15.44 per 1000 person-years, while it was 25.56 per 1000 person-years for those with NASH. Liverspecifc mortality among patients with NAFLD has been reported to be at a rate of 0.77 per 1000 person-years, while the rate is approximately 15 times higher among those

with NASH (11.77 per 1000 person-years). [9] Recent studies have shown that for patients with NAFLD and stage F0, the HR for overall mortality was 1.9 but increased to HR of 104.9 for those with F4 (cirrhosis) fbrosis and that those with F3–F4 fbrosis incurred a 3-fold higher overall mortality than those without liver disease. [102] A study conducted in 2017 found there were 184,905 deaths from liver complications due to NAFLD, which translated into an age standard rate (ASR) of NAFLD liver deaths of 2.32 per 100,000 persons. From 1990 to 2017, investigators using global data reported that the age-standardized DALY rate (ASDR) for cirrhosis-NAFLD also increased at an annual percentage change (APC) of 0.3% while the ASR for compensated and decompensated cirrhosis due to NASH increased more than for any other cause of cirrhosis (by 33.2% for compensated cirrhosis and 54.8% for decompensated cirrhosis) for the study period of 1990–2017. [92] HCC is an important complication of CLD and NAFLD and is now the third-leading cause of cancer death worldwide due to its very poor prognosis. [125] As a result, incidence and mortality rates are roughly equivalent. In 2018, the estimated global incidence rate of liver cancer per 100,000 person-years was 9.3, while the corresponding mortality rate was 8.5. [126] In patients with NAFLD, the annual incidence of HCC has been reported to be 1.8 cases per 1000 person-years with an overall mortality rate of 5.3 deaths per 1000 person-years. [80] Others have reported NAFLD increasing annually by 1.42% from 2012 to 2017, with the age-standardized death rate (ASDR) for NAFLD and liver cancer also increasing over the same period. [127] Similar reports have come from Asia, the Middle East and North Africa region where investigators studied the liver complications of cirrhosis and liver cancer. They reported that Asia accounted for 48.3% of the global incidence of NAFLD liver complications and for 46.2% of deaths attributable to NAFLD liver complications, while the MENA regions accounted for 8.9% of the global incidence of NAFLD and liver complications and 8.6% deaths attributable to NAFLD and liver complications. [128] HCC related to NAFLD and NASH in Japan is the most common malignancy and cause of death among patients with NAFLD/NASH with higher mortality observed among those with advanced disease (up to 40% in almost 3 years). [129] Prevention of NAFLD may be even more relevant as recent studies have found that HCC can develop in those with NAFLD without having developed fbrosis. [130] As noted previously, cardiovascular mortality is the most common cause of death among NAFLD. In this context, the presence of T2DM, very low-density lipids, hepatic overproduction of glucose, infammatory factors, C-reactive protein, coagulation factors and insulin resistance, which are commonly found among patients with NAFLD, can contribute to the increased risk of death from cardiovascular diseases. [131–133] It has been estimated that 5–10% of patients with NAFLD die from cardiovascular disease and have a 2-fold increase in risk for cardiovascular disease. Interestingly, recent data suggest that the stage of fbrosis may be associated with CVD even in the presence of cardiometabolic risk factors. [134] These data suggest that the underlying pathophysiology that hastens the development of fbrosis in NAFLD may also promote the development of CVD. [135–138] In fact, this may help explain recent fndings that NAFLD was not directly related to CVD mortality but rather was associated with both the prevalence and incidence of 15

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

coronary heart disease, atherosclerosis and hypertension, suggesting that the presence of NAFLD can indirectly increase the risk for CVD mortality through its affect on CVD related risk factors. [135] Findings from this study were further enhanced in a more recent meta-analysis where investigators found that those with NAFLD were at 1.5 times higher long-term risk for fatal or nonfatal CVD events, especially in those with advanced liver disease and higher fbrosis stages. [139] In addition to CVD and liver-specifc death, cancerrelated deaths are also increased among NAFLD. [140–142] In a recent meta-analysis, researchers determined that NAFLD was signifcantly intertwined with extrahepatic cancers. From their analysis, these investigators determined that the odds of developing colorectal cancer (CRC) in patients with NAFLD were almost doubled, while the odds for developing intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma in patients with NAFLD were increased 2.5 times compared to those without NAFLD. Furthermore, the odds of developing breast cancer gastric cancer, pancreatic cancer, prostate cancer and esophageal cancer were also found to be signifcantly increased. [141] These data suggest an association between NAFLD and extrahepatic malignancy and related mortality. Most recently, attention has been given to the association of sarcopenia, NAFLD and mortality. Sarcopenia is defned as the loss of muscle mass and strength, which has generally been associated with the elderly, although it is highly prevalent in persons with end-stage liver disease. The exact pathophysiologic mechanisms in which NAFLD and sarcopenia interact are still to be investigated; however, the current understanding is that the presence of insulin resistance, low vitamin D levels, infammatory myokines and physical inactivity, all present in NAFLD, contribute to increased proteolysis, myosteatosis, increased oxidative stress and decreased uptake of glucose in the muscle, which contributes to the development of sarcopenia. On the other hand, these factors are also present in those with sarcopenia, suggesting a shared pathway for the development of each. Although most studies to date have been carried out among persons from Asia, several studies showed that those with sarcopenia had a 2.3- to 3.3-fold increased risk of NAFLD and a 2-fold increased risk of fbrosis, independent of obesity or IR. [143–145] In this light, the presence of sarcopenia in those with NAFLD has also been linked with an almost 2 times higher risk for allcause mortality, a 3 times higher risk for cardiac-related mortality and 2 times higher risk for cancer-related mortality. [146] 2.9 CONCLUSIONS Due to the increasing global prevalence of obesity and T2DM, the prevalence of NAFLD and its associated adverse disease outcomes is increasing. The only proven treatments are weight loss and increased physical activity, which are hard to achieve and sustain, especially if the social determinants of health are not part of the comprehensive management plan. This increasing disease burden and limited treatment options are further compounded by lack of awareness among patients, health care providers and policy makers. It is only through a comprehensive multiprong approach that the challenge of NAFLD can be met at both the local and the global levels to change the trajectory of this important liver disease. 16

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102. Hagström H, Nasr P, Ekstedt M, Hammar U, Stål P, Hultcrantz R, Kechagias S. Fibrosis stage but not NASH predicts mortality and time to development of severe liver disease in biopsy-proven NAFLD. J Hepatol. 2017;67(6):1265–1273. 103. Vusirikala A, Thomas T, Bhala N, Tahrani AA, Thomas GN, Nirantharakumar K. Impact of obesity and metabolic health status in the development of non-alcoholic fatty liver disease (NAFLD): a United Kingdom population-based cohort study using the health improvement network (THIN). BMC Endocr Disord. 2020;20(1):96. 104. Golabi P, Otgonsuren M, de Avila L, Sayiner M, Rafq N, Younossi ZM. Components of metabolic syndrome increase the risk of mortality in nonalcoholic fatty liver disease (NAFLD). Medicine (Baltimore). 2018;97(13):e0214. 105. Stepanova M, Rafq N, Younossi ZM. Components of metabolic syndrome are independent predictors of mortality in patients with chronic liver disease: a population-based study. Gut. 2010;59(10):1410–1415. 106. Rodriguez-Hernandez H, Cervantes-Huerta M, Luis Gonzalez J, Dolores Marquez-Ramirez M, RodriguezMoran M, Guerrero-Romero F. Nonalcoholic fatty liver disease in asymptomatic obese women. The work was originated in the Biomedical Research Unit of the Mexican Social Security Institute at Durango, Mex. J Hepatol. 2010;9:144–149. 107. Jinjuvadia R, Antaki F, Lohia P, Liangpunsakul S. The association between nonalcoholic fatty liver disease and metabolic abnormalities in the United States population. J Clin Gastroenterol. 2017;51:160–166. 108. Burra P, Becchetti C, Germani G. NAFLD and liver transplantation: disease burden, current management and future challenges. JHEP Rep. 2020;2(6):100192. 109. Ahmed MH, Noor SK, Bushara SO, Husain NE, Elmadhoun WM, Ginawi IA, et al. Non-alcoholic fatty liver disease in Africa and Middle East: an attempt to predict the present and future implications on the healthcare system. Gastroenterology Res. 2017;10(5):271–279. 110. Kalia HS, Gaglio PJ. The prevalence and pathobiology of nonalcoholic fatty liver disease in patients of different races or ethnicities. Clin Liver Dis. 2016;20(2):215–224. 111. Messiah SE, Vidot DC, Baeker B, Jordan A, Kristopher L, Khorgami Z, De La Cruz-Muñoz N. Ethnic and gender differences in the prevalence of nonalcoholic steatohepatitis among bariatric surgery patients. Bariatr Surg Pract Patient Care. 2016;11(4). https://doi. org/10.1089/bari.2016.0018 112. Selvakumar PKC, Kabbany MN, Nobili V, Alkhouri N. Nonalcoholic fatty liver disease in children: hepatic and extrahepatic complications. Pediatr Clin North Am. 2017;64(3):659–675. 113. Kotronen A, Johansson LE, Johansson LM, et al. A common variant in PNPLA3, which encodes adiponutrin, is associated with liver fat content in humans. Diabetologia. 2009;52:1056–1060. 114. Chinchilla-López P, Ramírez-Pérez O, Cruz-Ramón V, Canizales-Quinteros S, Domínguez-López A, Ponciano-Rodríguez G, et al. More evidence for the genetic susceptibility of Mexican population to nonalcoholic fatty liver disease through PNPLA3. Ann Hepatol. 2018;17:250–255.

115. Martínez LA, Larrieta E, Kershenobich D, Torre A. The expression of PNPLA3 polymorphism could be the key for severe liver disease in NAFLD in hispanic population. Ann Hepatol. 2017;16:909–915. 116. Sookoian S, Pirola CJ. Meta-analysis of the infuence of I148M variant of patatin-like phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology. 2011;53:1883–1894. 117. Barbara M, Scott A, Alkhouri N. New insights into genetic predisposition and novel therapeutic targets for nonalcoholic fatty liver disease [Internet]. Hepatobiliary Surg Nutr. 2018;7:372–381. http://doi. org/10.21037/hbsn.2018.08.05 118. Sliz E, Sebert S, Würtz P, Kangas AJ, Soininen P, Lehtimäki T, et al. NAFLD risk alleles in PNPLA3, TM6SF2, GCKR and LYPLAL1 show divergent metabolic effects. Hum Mol Genet [Internet]. 2018;27:2214–2223. http://doi. org/10.1093/hmg/ddy124 119. Anstee QM, Day CP. The genetics of NAFLD [Internet]. Nat Rev Gastroenterol Hepatol. 2013;10:645–655. 120. Walker RW, Lê KA, Davis J, Alderete TL, Cherry R, Lebel S, Goran MI. High rates of fructose malabsorption are associated with reduced liver fat in obese African Americans. J Am Coll Nutr. 2012;31(5):369–374. 121. Mouzaki M, Loomba R. Insights into the evolving role of the gut microbiome in nonalcoholic fatty liver disease: rationale and prospects for therapeutic intervention. Therap Adv Gastroenterol. 2019;12:1756284819858470. http://doi. org/10.1177/1756284819858470. PMID: 31258623; PMCID: PMC6591661. 122. Leung C, Rivera L, Furness JB, Angus PW. The role of the gut microbiota in NAFLD. Nat Rev Gastroenterol Hepatol. 2016;13(7):412–425. http://doi.org/10.1038/ nrgastro.2016.85. Epub 2016 Jun 8. PMID: 27273168. 123. Aron-Wisnewsky J, Vigliotti C, Witjes J, Le P, Holleboom AG, Verheij J, Nieuwdorp M, Clément K. Gut microbiota and human NAFLD: disentangling microbial signatures from metabolic disorders. Nat Rev Gastroenterol Hepatol. 2020;17(5):279–297. http:// doi.org/10.1038/s41575-020-0269-9. Epub 2020 Mar 9. PMID: 32152478. 124. Schwimmer JB, Johnson JS, Angeles JE, Behling C, Belt PH, Borecki I, et al. Microbiome signatures associated with steatohepatitis and moderate to severe fbrosis in children with nonalcoholic fatty liver disease. Gastroenterology. 2019;157:1109–1122. 125. World Health Organization (WHO). World cancer mortality. Obtained from the world wide web at: www. who.int/news-room/fact-sheets/detail/cancer. Last accessed on 4/7/22. 126. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. 127. Paik JM, Golabi P, Biswas R, Alqahtani S, Venkatesan C, Younossi ZM. Nonalcoholic fatty liver disease and alcoholic liver disease are major drivers of liver mortality in the United States. Hepatol Commun. 2020;4(6):890–903. 128. Golabi P, Paik JM, AlQahtani S, Younossi Y, Tuncer G, Younossi ZM. Burden of non-alcoholic fatty liver

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disease in Asia, the Middle East and North Africa: data from Global Burden of disease 2009–2019. J Hepatol. 2021;75(4):795–809. http://doi.org/10.1016/j. jhep.2021.05.022. Epub 2021 May 31. PMID: 34081959. Eguchi Y, Wong G, Lee IH, Akhtar O, Lopes R, Sumida Y. Hepatocellular carcinoma and other complications of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in Japan: a structured review of published works. Hepatol Res. 2021;51(1):19–30. http:// doi.org/10.1111/hepr.13583. Epub 2020 Dec 9. PMID: 33091191. White DL, Kanwal F, El-Serag HB. Association between nonalcoholic fatty liver disease and risk for hepatocellular cancer, based on systematic review. Clin Gastroenterol Hepatol. 2012;10:1342–1359.e2. Targher G. Non-alcoholic fatty liver disease, the metabolic syndrome and the risk of cardiovascular disease: the plot thickens. Diabet Med. 2007;24:1–6. Bhatia LS, Curzen NP, Calder PC, Byrne CD. Non-alcoholic fatty liver disease: a new and important cardiovascular risk factor? Eur Heart J. 2012;33(10):1190–1200. http://doi.org/10.1093/eurheartj/ehr453. Epub 2012 Mar 8. PMID: 22408036. Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, et al. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology.2003;37:917–923. Taylor R, Taylor R, Bayliss S et al. Association between fbrosis stage and outcomes of patients with non-alcoholic fatty liver disease: a systematic review and meta-analysis. Gastroenteorlogy. 2020;20:30137. Wu S, Wu F, Ding Y, et al. Association of non-alcoholic fatty liver disease with major adverse cardiovascular events: a systematic review and meta-analysis. Sci Rep. 2016;6:1–14. Zhou Y, Zhou X, Wu S, et al. Synergistic increase in cardiovascular risk in diabetes mellitus with nonalcoholic fatty liver disease: a meta-analysis. Eur J Gastroenterol Hepatol. 2018;30:631–636. Stefan N, Häring H, Cusi K. Non-alcoholic fatty liver disease: causes, diagnosis, cardiometabolic consequences, and treatment strategies. Lancet Diabetes Endocrinol. 2019;7:313–324. Paik JM, Henry L, De Avila L, Younossi E, Racila A, Younossi ZM. Mortality related to nonalcoholic fatty liver disease is increasing in the United States. Hepatol Commun. 2019;3(11):1459–1471. http://doi.

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3 GENETICS OF NAFLD

3 Genetics of NAFLD Luca Valenti and Cristiana Bianco

CONTENTS 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2 Heritability of NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3 Genetic Architecture of NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3.1 Main Common Genetic Variants Associated with NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3.2 Other Inherited Risk Variants for NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.3.3 Role of Rare Genetic Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.4 Genetic and Environmental Factors: Two Sides of the Same Coin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.5 Clinical Applications: Precision Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Key Points ■

NAFLD is a complex trait, and the susceptibility to develop the disease depends on the interaction between inherited and environmental factors.

■

Variants in gene-regulating hepatic handling of lipid metabolism play a key role in the pathogenesis of hepatic fat accumulation and NAFLD.

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Genetic factors modulating hepatic fat drive the progression toward cirrhosis and hepatocellular carcinoma.

3.1 INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is now the most frequent liver disease, affecting about a quarter of the population globally, and it has become a leading cause of liver-related morbidity and mortality. The term includes a large spectrum of clinical entities, from simple steatosis to progressive steatohepatitis, cirrhosis and hepatocellular carcinoma (HCC). It is a complex trait, whose risk of onset and progression varies among individuals. Inherited factors, metabolic status (type 2 diabetes [T2D] and obesity, overall) and lifestyle contribute to disease susceptibility and the heterogeneity of clinical presentation. In the last 15 years, genome-wide association studies (GWAS), whole-exome sequencing (WES) and wholegenome sequencing (WGS) have allowed us to identify an initial set of genetic variants and deranged biological pathways implicated in the genetic susceptibility to NAFLD, improving the comprehension of disease pathophysiology. 3.2 HERITABILITY OF NAFLD Genetic predisposition (or susceptibility) can be defned as an increased likelihood of developing a disease based on a person’s genetic architecture. Common diseases are complex traits that depend on the interaction between genetic predisposition and environmental factors. According to the commonest view, several common genetic variants with a small individual impact on the phenotype contribute to the development of common traits, but more recent data suggest that rarer variants with a large effect size can contribute as well. Several research approaches support the notion that hepatic fat accumulation and NAFLD variability are strongly infuenced by inherited factors.1 First, twin studies showed that more than a half of the variability of DOI: 10.1201/9781003386698-4

hepatic fat content and circulating liver enzymes (such as alanine aminotransferase [ALT] levels) depends on heritability.2,3 From these studies, it also emerged that liver fat and hepatic fbrosis are shared traits, suggesting that hepatic fat accumulation has a causal role in progressive liver disease. More recent general population studies employing magnetic resonance imaging to quantify liver fat and elastometry to estimate liver fbrosis led to the same conclusion.2–4 Second, family studies showed that the risk of NAFLD and progressive liver disease is higher in relatives of patients affected by cirrhotic NAFLD as compared to the general population, independently of metabolic comorbidities.5 Lastly, multiethnic cohort studies demonstrated a marked interethnic variability in NAFLD susceptibility, which is higher in Hispanics, followed by East Asians, intermediate in Europeans and lower in African Americans independently of confounding factors.6 3.3 GENETIC ARCHITECTURE OF NAFLD 3.3.1 Main Common Genetic Variants Associated with NAFLD The mapping of the genetic architecture of common diseases started with the development and application of approaches based on GWAS, complemented by candidate gene association studies. Indeed, starting from large and well-characterized cohorts, GWAS permit the identifcation of the main common genetic variants associated with a trait (e.g., hepatic fat accumulation) or a condition (e.g., NAFLD). The idea is that susceptibility to complex diseases is accounted for by common genetic variants (with a minor allele frequency [MAF] ≥5%), each of which explains a small part of the risk in the general population. Studies conducted in recent years have allowed us to identify a handful of common genetic determinants of hepatic fat accumulation that contribute to explaining the interindividual and interethnic variability of this condition (Figure 3.1). Notably, most of the main genetic variants implicated in NAFLD pathogenesis affect proteins implicated in lipid metabolism, determining quantitative or qualitative changes of lipids within hepatic deposits (Table 3.1). The largest fraction of genetic predisposition to NAFLD and progressive liver disease is accounted for the rs738409 C>G single-nucleotide polymorphism (SNP), encoding for an aminoacidic substitution of methionine for isoleucine at position 148 (I148M protein variant) of patatin-like phospholipase domain containing3 (PNPLA3). The I148M 23

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

Table 3.1: List of Common and Rare Variants Associated with NAFLD Gene

Function

Variant

Impact on Protein

PNPLA3

Lipid droplets remodeling

rs738409 C>G

p.I148M

TM6SF2

VLDL secretion

rs58542926 C>T

p.E167K

MBOAT7

Lipid droplets remodeling, rs641738 C>T phospholipid metabolism GCKR Regulation of de novo rs1260326 T>C lipogenesis HSD17B13 Hepatic retinol metabolism ? rs72613567 T > TA

Linked to 3’-UTR p.P446L Alternate splicing

Effect of the Variant

Phenotype

Loss plus gain of function ↑NAFLD, NASH, fbrosis, HCC Loss of function ↑NAFLD, NASH, fbrosis, HCC Reduced expression ↑NAFLD, NASH, fbrosis, HCC Loss of function ↑NAFLD, NASH, fbrosis Loss of function ↓NASH, fbrosis, HCC

PPP1R3B

Glycogen synthesis

rs4841132 G > A

Gain of function

IFNL4

Innate immunity

Alternative protein

MERTK

Innate immunity, HSCs activation Lipoprotein remodeling

rs368234815 TT > dG Alternative protein translation site rs4374383 G > A Noncoding variant

↓NAFLD, NASH, fbrosis, HCC ↓NASH, fbrosis

Reduced expression

↓Fibrosis

rs236918 G > C

Noncoding variant

Gain of function

rs695366 G > A

Promoter variant

Gain of function

↑NAFLD, NASH, fbrosis ↓NASH

rs4880 C > T

Loss of function Loss of function

↑Fibrosis ↓NAFLD, fbrosis

p.E342K

Loss of function Loss of function Loss of function

↑Fibrosis ↑Fibrosis ↑Fibrosis ↑NAFLD, NASH, fbrosis, HCC ↑NAFLD ↑Fibrosis. HCC ↑NASH

PCSK7 UCP2

SOD2 MARC1

Mitochondrial lipid metabolism, oxidative phosphorylation Mitochondrial antioxidant Oxidative stress?

Lipoprotein remodeling Lipid synthesis Leptin signaling Adiposity and glucose metabolism, unclear HFE Iron metabolism CP Iron metabolism SERPINA1 Immunomodulation, ER stress APOB VLDL secretion

rs429358 T > C rs2792751 C > T rs12077210 C > T rs11858624 G > T

p.A16V p.M187K p.A165T p.C112R p.V43I Intronic variant p.P299H

rs1800562 G > A

p.C282Y

Several

Protein change

Loss of function

LIPA TERT ATG7

Several Several rs143545741 C>T rs36117895 T>C

Protein change

Loss of function Loss of function Loss of function

APOE GPAM LEPR PYGO1

Lipid catabolism Cellular senescence Autophagy

p.P426L p.V471A

variant predisposes to the whole spectrum of NAFLD, including development of steatohepatitis (NASH), progression to severe fbrosis-cirrhosis and HCC onset independently of fbrosis.7–9 Interestingly, the presence of the PNPLA3 variant is responsible for the 15–27% of the population attributable fraction of severe NAFLD, namely cirrhosis and HCC.10 PNPLA3 is a protein expressed on the surface of lipid droplets (LDs) with hydrolytic activity on glycerolipids, but it also has an acyltransferase activity, resulting in the remodeling of phospholipids and triglycerides (TGs) within LDs. The I148M variant induces a loss of function in protein’s enzymatic activity by reducing the substrate access to the enzyme’s active site; in addition, the mutated protein acquires the ability of evading ubiquitylation and avoiding proteasomal degradation. Thus the nonfunctional protein accumulates on LDs and inhibits the metabolization of TGs, promoting their storage and LDs enlargement.11,12 PNPLA3 is also 24

↓NAFLD ↑NAFLD ↑NAFLD, NASH ↓NAFLD

highly expressed in hepatic stellate cells (HSCs), where it is responsible for the hydrolysis of retinyl esters. Here, the loss of function induced by the I148M variant determines the retention of retinol and HSCs, as a response to chronic infammation, are activated to myofbroblast-like cells and secrete collagen, leading to liver fbrosis.11 The phenotypic expression of this variant is mainly promoted by obesity and insulin resistance. Indeed, PNPLA3 expression is induced by insulin through the activation of the transcription factor sterol regulatory element binding protein 1c (SREBP1c) and carbohydrate response element binding protein (ChREBP).12,13 The rs58542926 C>T in the transmembrane 6 superfamily member 2 (TM6SF2) is a missense mutation encoding for the E167K variant that has been associated with NAFLD, NASH and hepatic fbrosis.14,15 TM6SF2 protein localizes in the endoplasmic reticulum and ER-Golgi intermediate compartment, where it is involved in the secretion and/or

3 GENETICS OF NAFLD

Figure 3.1

Genetic loci involved in the pathophysiology of NAFLD

ABHD5: Abhydrolase domain containing 5; APOB: Apolipoprotein B; APOE: Apolipoprotein E; ATG7: Autophagyrelated-7; CP: Ceruloplasmin; FXR: Farnesoid X receptor; GCKR: Glucokinase regulator; GPAM: Glycerol-3-phosphate aminotransferase; HFE: Hemochromatosis gene; HSD17B13: 17-beta hydroxysteroid dehydrogenase 13; IFNL4: Interferon lambda 4; LEPR: Leptin receptor; LIPA: Lysosomal acid lipase; MARC1: Mitochondrial amidoxime reducing component 1; MBOAT7: Membrane bound O-acyl transferase 7; MERTK: Mer T kinase; PCSK7: Proprotein convertase subtilisin/ kexin 7; PCSK9: Proprotein convertase subtilisin/kexin 9; PNPLA3: Patatin-like phospholipase domain-containing 3; PPP1R3B: Protein phosphatase 1 regulatory subunit 3B; SERPINA1: Serpin family A member 1; SOD2: mitochondrial Superoxide dismutase; TERT: human Telomerase reverse transcriptase; TGF-b1: Transforming growth factor beta 1; TM6SF2: Transmembrane 6 superfamily member 2; UCP2: Uncoupling protein 2; VLDL: very-low-density lipoproteins. Genetic variants are classifed according to the biological mechanism by which the encoded proteins contribute to the development of liver disease. Green arrows indicate benefcial pathways, while red arrows indicate pathological process/lipid fuxes.

lipidation of very-low-density lipoprotein (VLDL). Indeed, some of the liver lipids deriving from de novo lipogenesis or absorption of remnant lipoprotein particles and fatty acids from the circulation are secreted back in the bloodstream in the form of VLDL. The variant induces a loss of function of the protein and thus impairs VLDL secretion, determines TGs accumulation and increases susceptibility to liver damage.16,17 Interestingly, the variant has a paradoxical effect in liver and heart; indeed, reducing circulating lipids confers a more favorable plasmatic lipid profle that provides protection against cardiovascular diseases.16 A number of studies have confrmed the impact of the variant rs641738 C>T in the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) gene on the full spectrum of NAFLD, including NASH and progression to advanced fbrosis,18 and on the risk of NAFLD-HCC development in patients without severe fbrosis19; recently, a new rare likely pathogenic variant has been identifed in patients with NAFLD-HCC.20 MBOAT7 encodes

lysophosphatidyl-inositol acyltransferase 1 (LPIAT1), an endoplasmic reticulum membrane protein involved in the remodeling of phospholipid acyl-chain in the so-called Land’s cycle21; the enzyme mediates the incorporation of arachidonic acid (AA) and other polyunsaturated fatty acids (PUFAs) into lysophosphatidyl-inositol (LPI) and other lysophospholipids. The rs641738 C>T variant induces a downregulation of MBOAT7 transcription and translation, resulting in a reduction of AA containing phospholipids and accumulation of LPI, which is converted in TGs that accumulate in LDs.22 In addition, it promotes the synthesis of phosphatidyl inositol and its degradation into diacylglycerol, fueling a vicious cycle of TGs generation and driving hepatic steatosis. On the other hand, some evidence suggest that downregulation of MBOAT7 itself can trigger hepatic fbrosis inducing proinfammatory and profbrotic cytokines via LPI accumulation.23,24 Glucokinase regulator (GCKR) is a protein that acts as an inhibitor of glucokinase (GCK) in response to glucose 25

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

levels; in fact, GCK is involved in the modulation of the infux of glucose in hepatocytes in response to insulin and activates glucose storage pathways including de novo lipogenesis and glycogen synthesis.25 The common missense SNP rs1260326 C>T, encoding the P446L variant of GCKR, lacks the ability to inhibit glucokinase in response to fructose-6-phosphate, and therefore the hepatic uptake of glucose is constitutively activated.26 The consequence is a decrease in fasting glycemia and insulin resistance, but at the same time glycolysis, de novo lipogenesis and hepatic steatosis are promoted.25,27 More recently, loss of function variants in the hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13), such as rs143404524 and rs72613567, have been identifed as protective factors against liver damage28,29; in particular, the rs72613567:TA encodes for a prematurely truncating and unstable protein with reduced enzymatic activity. The HSD17B family consists of catalytic enzymes acting toward 7β-hydroxy and -keto steroid substrates; most HSD17B family members are involved in the activation/ inactivation of sex hormones. HSD17B13 is mainly expressed in the liver and localized on the surface of LDs in hepatocytes, where it exerts a retinoic dehydrogenase activity promoting the conversion of retinol to retinoic acid.29 Although the molecular mechanisms have not been well-uncovered yet, it is known that the effect of these variants is not accounted for by protection against hepatic fat accumulation but involves direct modulation of infammation and fbrogenesis. 3.3.2 Other Inherited Risk Variants for NAFLD The genetics of NALFD is an ever changing feld, and the list of genetic variants is increasing day by day. The rs4841132 G>A variant in the protein phosphatase 1 regulatory subunit 3B (PPP1R3B) has been identifes as a protective factor against hepatic fat accumulation modulating hepatic lipid metabolism.30 Polymorphisms in genes that regulate innate immunity, fbrogenesis and oxidative stress may modify the effect of fat accumulating in hepatocytes. For example, variants in the interferon lambda 3/4 (IFNL4) locus encoding for the alternative interferon lambda-3 and lambda-4 proteins modulate the activation of innate immunity and necroinfammation.31 The rs4374383 G>A in Mer tyrosine kinase (MERTK) reduces MERTK expression in hepatic myeloid cells and therefore the activation of HSCs, protecting from hepatic infammation and fbrogenesis.32 Conversely, the rs236918 G>C in proprotein convertase subtilisin/kexin type 7 (PCSK7) seems to be linked to lipid and iron metabolism and the promotion of fbrogenesis.33 Variants in uncoupling protein 2 (UCP2) and mitochondrial manganese-dependent superoxidase dismutase 2 (SOD2), respectively the rs695366 G>A and the rs4880 C>T, infuence fatty acid oxidation and oxidative stress in mitochondria, 34,35 whereas the p.M187K variant in mitochondrial amidoxime reducing component 1 (MARC1) has a protective role against disease progression and cirrhosis.36 More recent studies highlighted variants in apolipoprotein E (APOE) and glycerol-3-phosphate aminotransferase (GPAM), which regulate hepatic lipid metabolism, in the leptin receptor (LEPR), involved in the modulation of appetite and fbrogenesis and in pygopus homolog-1 (PYGO1), as risk factors for NAFLD and cirrhosis progression.37,38 In this list, other genes known to cause other inherited diseases should be added. Indeed, genetic variants known 26

to infuence iron metabolism (HFE C282Y, responsible for hereditary hemochromatosis) have been associated with more severe liver damage in patients with NAFLD, probably due to the predisposition to iron accumulation in hepatocytes.39 Similarly, variations in the ceruloplasmin gene (CP) have been associated with increased hepatic iron stores and more advanced fbrosis in patients with NAFLD.40 In addition, the PiZ variant in SERPINA1 (responsible for alpha-1-antitrypsin defciency) seems to impair lipid secretion and play a role in fat-associated liver damage.41 3.3.3 Role of Rare Genetic Variants Genetic variants are defned as “rare” when present in the general population with a minor allele frequency (MAF) of less than 1%. There is initial evidence that rare missense or nonsense genetic variants with a strong impact on protein structure and function predispose with a large effect size to hepatic fat accumulation and liver damage.20 These variants in key genes regulating hepatic lipid metabolism are responsible for disease clustering in families. As example, rare mutations on apolipoprotein B (APOB), responsible for familial hypobetalipoproteinemia, predispose to steatosis and severe progressive liver diseases because of the lipid compartmentalization in hepatocytes due to the inability to secrete VLDL.42 Moreover, APOB mutations predispose to NAFLD-HCC.20 APOB is also involved in chylomicrons secretion and may cause damage to the intestinal barrier due to lipid accumulation on enterocytes and malabsorption of liposoluble vitamins. It should be remembered that severe genetic disorders may manifest with NAFLD even in children, as in the case of mutations of LIPA; these are responsible for lysosomal acid lipase defciency and cause the accumulation of triglycerides and cholesteryl esters in hepatocytes.43 Another key mechanism involved in the progression of liver disease is cell senescence related to telomerase shortening. Indeed, loss-of-function variants in telomerase reverse transcriptase (TERT) have been associated with progressive liver disease and HCC development.44,45 Finally, there is evidence that rare loss-of-function variants in autophagy-related-7 (ATG7) and the low-frequency hypomorphic V471A variant predispose to progressive NAFLD by impairing the autophagic fux, leading to the accumulation of fat and of the sequestome protein p62 in damaged hepatocytes, thereby triggering ballooning and hepatic infammation.46 3.4 GENETIC AND ENVIRONMENTAL FACTORS: TWO SIDES OF THE SAME COIN The phenotypic expression of complex traits, as NAFLD is, depends on the interaction between genetic background and environmental determinants. The magnitude of the effect of a given variant may be modulated by the number of alleles of another genetic variant (gene–gene interaction) or by an environmental factor (gene–environment interaction). At the general population level, being carriers of common genetic risk variants is not suffcient to develop NAFLD or progressive liver disease. The main environmental trigger of NAFLD is represented by adiposity, which is related to insulin resistance and hyperinsulinemia.47,48 Indeed, the impact of genetic variants, as for example PNPLA3 I148M, on hepatic fat and predisposition to cirrhosis increases with body mass index (BMI), being higher in obese and very obese individuals.13 Similarly,

3 GENETICS OF NAFLD

Figure 3.2

Inherited and acquire.d factors involved in the development and progression of NAFLD

BMI: Body mass index; GCKR: Glucokinase regulator; HCC: Hepatocellular carcinoma; MBOAT7: Membrane-bound O-acyl transferase 7; PNPLA3: Patatin-like phospholipase domain-containing 3; TM6SF2: Transmembrane 6 superfamily member 2; T2D: type 2 diabetes.

an interaction between BMI and TM6SF2 E167K, GCKR P446L, and rare ATG7 variants as well, but not with other BMI-associated phenotypes such as TGs levels, has been observed in a population cohort study.13 The development of the disease in obese individuals is related to the concomitant insulin resistance and hyperinsulinemia, that for example amplify the PNPLA3-associated genetic risk of NAFLD.49 Similarly, the susceptibility to NAFLD linked to PNPLA3 is exacerbated by the consumption of industrial fructose in soft drinks, but the effect of genetic predisposition is positively modulated by a Mediterranean dietary pattern and physical activity.50,51 In addition, high insulin levels induce downregulation of MBOAT7, and in the case of insulin resistance, this process promotes hepatic lipogenesis and steatosis, particularly in carriers of genetic risk variants.22 PNPLA3 offers the clearest example of the effect of gene–gene interaction in NAFLD pathogenesis. An overview of the interaction between genetic and acquired factors involved in the development and progression of NAFLD is shown in Figure 3.2. 3.5 CLINICAL APPLICATIONS: PRECISION MEDICINE The main focus of precision medicine is the individualized management of the disease from disease screening to follow-up strategy and treatment. Genetic predisposition has been often called upon for the development of a personalized health care approach. Mendelian randomization studies that exploited as instruments the genetic risk variants for NAFLD just presented have provided data supporting the notion that hepatic fat accumulation is not an innocent bystander but a key driver of progressive liver disease, fbrosis and HCC.

Importantly, NAFLD-HCC develops in almost a half of noncirrhotic patients.52 Identifying patients who may beneft from cost-effective surveillance program therefore remains a major challenge, and noninvasive tools able to discriminate individuals at risk of liver disease progression are urgently needed. A precision medicine approach using the knowledge emerging from human genomics partly meets this need. Although the genotyping of a risk variant is a relatively simple and cheap investigation, no single SNP permits an adequate risk stratifcation in complex traits, and the use of single variants (e.g., the variant with the higher impact in the natural history of the disease, PNPLA310) for this purpose is not suffciently accurate.53 However, larger panels of genetic determinants included in polygenic risk scores (PRSs) could be a sensible approach. PRSs capture the individual’s genetic predisposition to develop a trait or an associated outcome and are calculated by summing the number of trait-associated alleles carried by an individual, which can be weighted by their effect size on the trait.54 Contrary to imaging, circulating biomarkers or other favoring comorbidities that represent a punctual assessment of disease predisposition, PRSs capture the potential to develop the outcome during the whole lifetime in the presence of triggering factors and may be used for screening purposes, especially in younger individuals and for conditions amenable to treatment. In the case of NAFLD, evidence supporting the possible utility of PRS is emerging. However, the diagnostic accuracy and the prognostic performance of these tools should be established in larger and prospective cohorts. Weighted PRS combining genetic loci, previously associated with liver damage, oxidative stress and fbrogenesis, 27

SECTION I: MECHANISMS OF NAFLD DEVELOPMENT

and common steatogenic risk variants, predict NAFLD and the full spectrum of liver damage in children and adults.55 This is particularly relevant when HCC risk has to be evaluated. Slightly less than one NAFLD patient in two develops HCC in a noncirrhotic liver, and surveillance is not recommended in the absence of liver cirrhosis, resulting in a delayed diagnosis and the worsening of prognosis due to limited treatment options. Thus PRSs based on genetic variants associated with NAFLD-HCC onset may be useful tools to improve the stratifcation of the risk. Some weighted and unweighted scores suitable for this purpose have already been proposed,56,57 and including common and rare variants identifed by NGS in a model of acquired clinical factors may improve the ability to discriminate the disease and correctly reclassify a considerable number of patients.20 There is still a lack of drug therapies approved for NAFLD. Recent genetic discoveries may assist in identifying molecular pathways that predispose to the disease and in targeting variant proteins that play a role in the development of progressive NAFLD in subsets of patients with distinct disease genetics and pathophysiology. Besides, this approach has been successfully used in some forms of cancer. Although now there is consistent evidence about the robust correlation between common genetic variants and NAFLD, knowledge about the implicated metabolic, epigenetic and biological mechanisms is still uneven.58 3.6 CONCLUSION Human genetics offers a privileged point of view on NAFLD. Identifcation of common and rare genetic risk variants has strengthened the understanding of the mechanisms at the base of the heterogeneous presentation of the disease. Furthermore, it has spurred the study of several pathogenic pathways implicated in hepatic fat accumulation, infammation, fbrogenesis and carcinogenesis. This knowledge may eventually impact on clinical practice, through enabling a medicine approach based on improved individual risk stratifcation and the development of targeted therapies. REFERENCES 1. Eslam M, Valenti L, Romeo S. Genetics and epigenetics of NAFLD and NASH: Clinical impact. J Hepatol. 2018;68(2):268–279. http://doi.org/10.1016/j. jhep.2017.09.003 2. Loomba R, Rao F, Zhang L, et al. Genetic covariance between γ-glutamyl transpeptidase and fatty liver risk factors: Role of β2-adrenergic receptor genetic variation in twins. Gastroenterology. 2010;139(3):836–845.e1. http://doi.org/10.1053/j.gastro.2010.06.009 3. Makkonen J, Pietiläinen KH, Rissanen A, Kaprio J, YkiJärvinen H. Genetic factors contribute to variation in serum alanine aminotransferase activity independent of obesity and alcohol: A study in monozygotic and dizygotic twins. J Hepatol. 2009;50(5):1035–1042. http:// doi.org/10.1016/j.jhep.2008.12.025 4. Loomba R, Schork N, Chen CH, et al. Heritability of hepatic fbrosis and steatosis based on a prospective twin study. Gastroenterology. 2015;149(7):1784–1793. http://doi.org/10.1053/j.gastro.2015.08.011 5. Long MT, Gurary EB, Massaro JM, et al. Parental non-alcoholic fatty liver disease increases risk of non-alcoholic fatty liver disease in offspring. Liver Int. 2019;39(4):740–747. http://doi.org/10.1111/liv.13956

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47. Valenti L, Bugianesi E, Pajvani U, Targher G. Nonalcoholic fatty liver disease: Cause or consequence of type 2 diabetes? Liver Int. 2016;36(11):1563–1579. http://doi.org/10.1111/liv.13185 48. Dongiovanni P, Stender S, Pietrelli A, et al. Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver. J Intern Med. 2018;283(4):356–370. http://doi.org/10.1111/joim.12719 49. Barata L, Feitosa MF, Bielak LF, et al. Insulin resistance exacerbates genetic predisposition to nonalcoholic fatty liver disease in individuals without diabetes. Hepatol Commun. 2019;3(7):hep4.1353. http://doi.org/10.1002/ hep4.1353 50. Nobili V, Liccardo D, Bedogni G, et al. Infuence of dietary pattern, physical activity, and I148M PNPLA3 on steatosis severity in at-risk adolescents. Genes Nutr. 2014;9(3). http://doi.org/10.1007/s12263-014-0392-8 51. Ma J, Hennein R, Liu C, et al. Improved diet quality associates with reduction in liver fat, particularly in individuals with high genetic risk scores for nonalcoholic fatty liver disease. Gastroenterology. 2018;155(1):107–117. http://doi.org/10.1053/j. gastro.2018.03.038 52. Piscaglia F, Svegliati-Baroni G, Barchetti A, et al. Clinical patterns of hepatocellular carcinoma in nonalcoholic fatty liver disease: A multicenter prospective study. Hepatology. 2016;63(3):827–838. http://doi. org/10.1002/hep.28368

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53. EASL–EASD–EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016;64(6):1388–1402. http://doi.org/10. 1016/j.jhep.2015.11.004 54. Wand H, Lambert SA, Tamburro C, et al. Improving reporting standards for polygenic scores in risk prediction studies. Nature. 2021;591(7849):211–219. http://doi. org/10.1038/s41586-021-03243-6 55. Bianco C, Tavaglione F, Romeo S, Valenti L. Genetic risk scores and personalization of care in fatty liver disease. Curr Opin Pharmacol. 2021;61:6–11. http://doi. org/10.1016/j.coph.2021.08.014 56. Gellert-Kristensen H, Richardson TG, Davey Smith G, Nordestgaard BG, Tybjærg-Hansen A, Stender S. Combined effect of PNPLA3, TM6SF2, and HSD17B13 variants on risk of cirrhosis and hepatocellular carcinoma in the general population. Hepatology. 2020;72(3):845–856. http://doi.org/10.1002/hep.31238 57. Bianco C, Jamialahmadi O, Pelusi S, et al. Non-invasive stratifcation of hepatocellular carcinoma risk in non-alcoholic fatty liver using polygenic risk scores. J Hepatol. 2021;74(4):775–782. http://doi.org/10.1016/j. jhep.2020.11.024 58. Sookoian S, Pirola CJ. Precision medicine in nonalcoholic fatty liver disease: New therapeutic insights from genetics and systems biology. Clin Mol Hepatol. 2020;26(4):461–475. http://doi.org/10.3350/ cmh.2020.0136

4 MECHANISMS OF HEPATOCYTE INJURY AND INFLAMMATION IN NAFLD

4 Mechanisms of Hepatocyte Injury and Inflammation in NAFLD Gopanandan Parthasarathy and Harmeet Malhi

CONTENTS 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 4.2 Lipotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 4.3 Infammasome Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.4 Genetic Modifers of Lipotoxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.5 Crosstalk with Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.5.1 Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.5.2 Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.5.3 T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.5.4 NK Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.5.5 B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.6 Interorgan Crosstalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.6.1 Gut–Liver Crosstalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.6.2 Adipose-Liver Crosstalk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 4.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Key Points ■

Bioactive and signaling lipids, such as saturated fatty acids, cholesterol and sphingolipids are hepatotoxic by activating organelle stress responses and proapoptotic signaling.

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Hepatocyte injury and death activate a cascade of infammatory and reparative responses such as infammasome activation, release of soluble mediators such as cytokines and secretion of extracellular vesicles.

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Genetic variants that infuence lipid droplet dynamics and lipid metabolism modulate human NASH—for example, variants in PNPLA3, TM6SF or MBOAT7 exacerbate, while HSD17B13 modulates liver injury.

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The liver is rich in immune cells, and a complex network of immune-mediated liver injury is described in NASH. Innate immune cells such as macrophages are particularly important and mediate crosstalk with hepatocytes, other immune cells and stellate cells, thus amplifying infammation, fbrosis and carcinogenesis.

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NASH occurs in the context of disordered organismal metabolism and infammation, in that the liver communicates with the gut and adipose tissue in the form of metabolites, PAMPs, DAMPs, extracellular vesicles and immune cells, which create feed-forward loops of metabolic dysregulation and chronic infammation.

4.1 INTRODUCTION The existing defnitions of nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) rely on hepatocellular injury and infammation as defning features. The focus of this chapter is the mechanisms that lead to injury and infammation. Steatosis (discussed in other chapters) is largely dependent on increased delivery of free fatty acids (FFA) to the liver due to enhanced adipose tissue lipolysis in insulin resistance (IR), diet-derived FFA and de novo lipogenesis, which is in part driven by dietary sugars1. Mechanistic insights in NASH have been obtained from cellular and animal model systems (Figure 4.1) with correlative studies in human liver samples. These studies have DOI: 10.1201/9781003386698-5

demonstrated the key role of ectopically accumulated lipids in activating cell death pathways and organelle stress, whether mitochondrial, endoplasmic reticulum (ER) or lysosomal stress. It is also clear from accumulated data and unsurprising given the complex immune composition of the liver, that hepatocyte–immune cell crosstalk plays an important role in the sterile infammatory response associated with NASH. The immune response in the liver is further modifed by interorgan crosstalk, such as gut–liver and adipose tissue–liver crosstalk. In this chapter, we provide a synopsis of these injury and infammation pathways. 4.2 LIPOTOXICITY The concept of lipotoxicity was introduced in the context of the obesity-associated type 2 diabetes mellitus and rapidly expanded to the liver with the recognition that several lipid classes, including saturated FFA, lysophosphatidyl cholines (LPCs), cholesterol and sphingolipids (ceramides and sphingosine 1-phosphate, S1P) were deleterious in NASH by inducing organelle stress and activating cell death pathways2. The saturated FFA, palmitate (Figure 4.2) and stearate, are directly toxic to hepatocytes via activation of proapoptotic signaling3. Monounsaturated fatty acids (MUFA) such as oleate and palmitoleate are not directly toxic and may mitigate the toxicity of saturated FFA by increasing their partitioning into neutral triglycerides3. In fact, knockout (-/-) of stearoyl-CA desaturase-1 (SCD1) decreased hepatocyte steatosis but increased SFA-induced apoptosis, injury and infammation4. Palmitate can drive the generation of LPC by phospholipase A2 and ceramides via de novo synthesis, thus it can be toxic directly or via lipid intermediaries5,6. LPC and ceramides can be generated by additional pathways, lecithin cholesterol acyltransferase and sphingolipid salvage pathway, respectively, which may also contribute to lipotoxicity. This interconnectedness of lipids may also explain why many of the same mechanistic pathways are activated by bioactive lipids. Crystals of free cholesterol, a distinct lipid class, are detected in patients with fbrosing NASH, and cholesterol content of mouse diets correlates with the fbrogenic potential of specifc diets, suggesting a role for cholesterol toxicity in progressive NASH7. 31

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Figure 4.1

Mouse models recapitulate NASH histology

Representative images from liver sections from a mouse model of diet-induced NASH stained with hematoxylin and eosin (panel A) and picro-sirius red (panel B). (A) The arrows denote the features of NASH: macrovesicular steatosis (solid black arrow), microvesicular steatosis (dashed black arrow), ballooned hepatocyte (blue arrow), lobular infammation (yellow arrow). (B) Pericellular fbrosis (solid black arrow).

Figure 4.2 Palmitate activates the intrinsic and extrinsic apoptosis pathways in hepatocytes (This fgure was created with BioRender.com.) Excess palmitate activates organelle stress in many forms including lysosomal permeabilization, endoplasmic reticulum (ER) stress, and autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). These processes lead to the activation of c-jun N-terminal kinase (JNK), induction of C/EBP homologous protein (CHOP) and upregulation of proapoptotic proteins PUMA and Bim (intrinsic pathway), leading to mitochondrial outer membrane permeabilization (MOMP). JNK and CHOP also lead to the transcriptional upregulation of TRAIL-R2. Ligand independent oligomerization of TRAIL-R2 activates caspase 8 leading to the cleavage of Bid to tBid (extrinsic pathway) and MOMP.

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4 MECHANISMS OF HEPATOCYTE INJURY AND INFLAMMATION IN NAFLD

SFAs and LPC activate proapoptotic signaling in hepatocytes via the mixed lineage kinase 3, c-Jun N-terminal kinase (JNK), and ER stress-induced induction of the proapoptotic transcription factor, C/EBP homologous protein (CHOP)2,3,8. These stress kinase and ER stressinduced pathways converge on mitochondria, and, indeed, SFAs shift the balance of proapoptotic and antiapoptotic Bcl-2 family proteins toward cell death, by increasing the expression of PUMA and Bim and inhibiting Mcl-1. Epigenetic mechanisms also regulate sensitivity to apoptosis as demonstrated by the microRNA, miR-296–5p, which inhibits the expression of PUMA and is inversely related to PUMA mRNA levels in NASH liver biopsies. Activation of autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1) also contributes to an increase in the levels of PUMA and Bim with sensitization to apoptosis. Bim is also upregulated by phosphatase-driven dephosphorylation and activation of the transcription factor, FOXO3a in palmitate-treated hepatocytes. SFAs also induced the expression of death receptors and sensitize hepatocytes to death receptor-induced apoptosis. Palmitate induces the upregulation of the death receptor, TRAIL-R2, and leads to clustering and ligand-independent activation of TRAIL-R2 by changing the organization of its plasma membrane domains. Increased membrane rigidity in SFA-treated hepatocytes is also implicated in organelle stress, such as lipotoxic activation of the unfolded protein response sensors, IRE1α and PERK. Recent studies have demonstrated that hepatocytes release proinfammatory extracellular vesicles with distinct signaling cargoes as a response to lipotoxic endoplasmic reticulum stress9. Changes in membrane fuidity are also impacted by cholesterol2,7. In hepatocytes, increased accumulation of cholesterol in intracellular membranes leads to increased membrane rigidity and organelle stress. Impaired activity of sarcoplasmic-endoplasmic reticulum calcium ATPase by increases in ER membrane cholesterol content lead to ER stress and activation of the unfolded protein response. A reduction in mitochondrial 2-oxoglutarate carrier function due to increases in mitochondrial membrane cholesterol result in mitochondrial glutathione depletion. In macrophages, the accumulation of free cholesterol may activate the infammasome (discussed later in the chapter). Ceramides are mechanistically linked to the development of IR. In addition, sphingosine 1-phosphate (S1P) is proinfammatory in the liver by recruiting monocyte-derived macrophages into the liver. Inhibitors of S1P receptors, such as FTY720, demonstrate a reduction in liver injury and infammation in preclinical models10. Thus, many bioactive lipid species are directly or indirectly deleterious to the liver by activating cellular stress responses, activating infammatory pathways or leading to cell death3. Mouse models that are defcient in pathways activated by lipotoxic signaling demonstrate attenuated NASH, such as mice defcient in the death receptor, TRAIL-R2, hepatocyte caspase 8 defcient or Bid silenced mice11. Markers of apoptosis such as apoptotic hepatocytes, cleaved caspases and death receptors are elevated in NASH human liver samples. Readouts of lipotoxic pathways may also serve as disease biomarkers in NASH. In this regard, hepatocyte-derived extracellular vesicles, the S1P content of extracellular vesicles and the caspase cleaved fragment of cytokeratin 18 (CK18 M30) have demonstrated utility in tracking NASH activity12. Lastly, several therapeutic agents that target various aspects of lipid homeostasis are in development. These include inhibitors

of acetyl coenzyme A carboxylase mediated de novo lipogenesis, diacylglycerol transferase which catalyzes triglyceride synthesis, agonists of PPARs which modulate lipid utilization and fux, and FXR agonists which regulate lipid metabolism13. 4.3 INFLAMMASOME ACTIVATION Infammasomes are cytosolic multiprotein complexes assembled downstream of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMP) binding to their respective pattern recognition receptors (PRRs). The canonical infammasomes are multiprotein complexes containing PRRs such as NODlike receptor (NLR) family members (NLRP1, NLRP3 and NLRC4), procaspase 1 and the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Downstream of PAMPs binding to toll-like receptors (TLRs), in a process called priming, the activation of the transcription factor nuclear factor κB (NF-κB) leads to production of cytokine precursors prointerleukin (IL)-1β and pro-IL-18. Proteolytic cleavage of procaspase 1 in the infammasome generates caspase-1, which in turn is necessary for the proteolytic cleavage of these precursors into interleukin (IL)-1β and IL-18. Caspase 1 activation also leads to an infammatory cell death, termed “pyroptosis,” via cleavage-mediated activation of the membrane poreforming protein gasdermin D (GSDMD). Association of the infammasome component NLRP3 with the metabolic syndrome is well established in various cell types in the liver and adipose tissue14. In liver biopsies from patients with NAFLD, elevated mRNA levels of NLRP3 components and GSDMD expression were correlated with NASH severity15,16. Experimental evidence in mouse models of NASH described lipotoxicity-induced upregulation of NLRP3 infammasome components at the transcriptional but not protein level. Conversely, in mice lacking NLRP3, ASC and caspase 1–infammasome components, diet-induced NASH was attenuated17. NLRP3 and NLRP6 infammasome defciency in mice led to altered gut microbiota composition and greater infux of TLR4 and TLR9 agonists into the portal circulation, thus worsening NASH18. In NASH mice, both hepatic and macrophage AIM2 expression was increased, mediated by increased TLR9 ligands19. Ligands implicated in infammasome activation include palmitate via TLR2, extracellular ATP via its cell membrane receptor–purinergic 2X7 receptor (P2X7R), cholesterol crystals, bile acids via their surface receptor TGR5 or their nuclear receptor FXR, ER stress, mitochondrial oxidative stress and mitochondrial DNA19–22. Conceivably, several approaches to target infammasomes as therapeutic intervention in NASH have been attempted. Sulforaphane, a small molecule inhibitor of NLRP3 infammasome activation attenuated diet-induced murine NASH23. Similarly, a P2RX7 inhibitor attenuated liver infammation and fbrosis in a nonhuman primate model of NASH22. Thus infammasome activation represents a crucial pathway in the pathogenesis of NASH and provides a link between cell death and infammation. Further exploration of therapeutic targeting of the infammasome in a cell type-specifc manner is warranted. 4.4 GENETIC MODIFIERS OF LIPOTOXICITY NASH pathogenesis involves a complex interplay of genetic and environmental factors. In the context of hepatocyte injury and liver infammation, the functional mechanistic contribution for how the genetic variants may increase risk 33

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for liver injury or infammation is known for a few of the many genetic risk factors. The patatin-like phospholipase domain-containing 3 (PNPLA3) gene encodes a protein associated with lipid droplets and has lipase activity. PNPLA3 I148M variant increases steatosis, fbrosis and HCC risk24. Lipotoxicity may be increased due to the I148M variant as it can inhibit the activity of adipose tissue triglyceride lipase. The TM6SF E167K variant impairs VLDL lipidation with accumulation of triglyceride and potentially other toxic lipids in the liver25. Glucokinase regulator (GCKR) P446L variant is a loss of function variant in which the ability to inhibit glucokinase is lost with an increase in de novo lipogenesis leading to increased hepatic steatosis. A genetic variant near membrane bound O-acyltransferase domain containing 7 (MBOAT7) is also associated with hepatic steatosis and fbrosis26. On the other hand, hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) loss-of-function variants (rs143404524 and rs72613567) are associated with a reduction in liver injury, suggesting that silencing or inhibiting HSD17B13 may be a potential therapeutic strategy for NASH27. While many of the reported variants are lipid droplet-associated proteins or impact lipid fux in the liver, it is not known whether these variants directly impact known toxic lipid mediators or shift lipid accumulation or fux to less harmful lipids. The variants could also, at a cellular level, shift the compartmental distribution of lipids, another potential area for future studies. 4.5 CROSSTALK WITH IMMUNE CELLS The liver contains many innate and adaptive immune cells that uniquely interface with the cellular compartments of the liver due to the microanatomical architecture of hepatic sinusoids. Two-thirds of hepatic blood supply is from the portal circulation; the immune-cell rich hepatic microenvironment serves as a crucial immunologic frewall against gut-derived signals, including pathogens, PAMPs and DAMPs in both health and disease. Long-liver resident cells include Kupffer cells (KCs), CD8+ tissue resident memory T (TRM) cells and type 1 innate lymphoid cells (ILC1s), but other cell types circulate through the liver and serve a patrolling role, including natural killer (NK) cells, γδ T cells, CD4+ and CD8+ αβ T cells, monocytes, B cells, invariant NKT (iNKT) cells, mucosal-associated invariant T (MAIT) cells and dendritic cells (DCs). Intrahepatic blood fow in the portal to central direction constructs a gradient of nutrients, oxygen, gut derivatives, and, in response to these, a zonation of immune cells. Mechanistically, because of hepatocyte lipotoxicity and apoptosis, release of soluble mediators such as the DAMP, high-mobility group box 1 (HMGB1), FFA, PAMPs, and extracellular vesicles leads to recruitment and infammatory activation of circulating immune cells (Figure 4.3). Subsequently, immune cells directly promote infammation by secreting infammatory cytokines including tumor necrosis factor-alpha and IL-1β but also promote a feed-forward loop of inciting hepatocyte injury, death, and further activation of other immune cells. Lastly, immune-mediated activation of hepatic stellate cells (HSCs) promotes fbrogenesis in NASH28. Alternatively, injury resolution models demonstrate that immune cells are also key for the termination of infammation, regression of fbrosis and hepatocyte regeneration. Given the multicellular and context-specifc roles of immune cells in NASH, we do not yet have an integrated understanding of the intrahepatic crosstalk, including spatiotemporal aspects and mechanistic insights; however, rapid advances 34

in single-cell multiomics and spatial analytics are expected to revolutionize our understanding of this crosstalk. 4.5.1 Macrophages Macrophages are the most abundant immune cell type in the liver and can be classifed into resident embryonic yolk sac-derived KCs and recruited bone marrow-derived macrophages (BMDMs), both of which are capable of self-renewal29. They have been extensively studied as a driver of NASH pathogenesis in human disease as well as animal models. In NASH, a lipotoxic environment impairs the survival and self-renewal of KCs30, and this niche is repopulated by BMDMs, which do not fully recapitulate homeostatic KC functions and are considered more proinfammatory. The resulting intrahepatic macrophage population is heterogeneous, and many subsets have been characterized, including premonocyte-derived KCs, monocyte-derived KCs and hepatic lipid-associated macrophages (LAMs)31. LAMs are characterized by Trem2 and Cd9 expression, enriched in fbrotic zones, and possess differential lipid handling capabilities. A similar NASHassociated macrophage (NAM) subset has also been described, reminiscent of LAMs in adipose tissue31, as well as scar-associated macrophages (SAM) in fbrotic human livers31. Microanatomical niche-derived signals that epigenetically educate macrophage identity and function have been described, for example reprogramming of liver X receptor (LXR) functions is implicated in promoting the Trem2+ macrophage phenotype31. Other examples implicated in macrophage activation include the transmembrane protein 173 (TMEM173 or STING) pathway32, toll-like receptor 4 (TLR4)33 and extracellular vesicles that mediate monocyte adhesion and recruitment34. Macrophages play a role in various stages of NASH by directly amplifying (1) steatosis by secretion of IL-1β, (2) infammation by chemokines and cytokines, including tumor necrosis factor alpha (TNF-α) and IL-1β, (3) monocyte recruitment by chemokines such as CCL235, and (4) fbrosis by activating hepatic stellate cells31. 4.5.2 Dendritic Cells Dendritic cells are recognized for their role in inducing the innate immune system by presenting antigens to and infammatory reprogramming of T cells, but they can also directly participate in the infammatory response, downstream of pattern recognition receptors. Although dendritic cells represent a relatively rare population in the liver, constituting less than 5% of immune cells, all 3 subsets, plasmacytoid (pDCs) and conventional (cDC1 and 2), have been associated with NASH but with differential effects on NASH pathogenesis. Human transcriptomic study suggested that cDC2 correlated with NASH severity while cDC1 were protective in NASH36, and, indeed, mice genetically defcient in cDC1 had worse diet-induced NASH37. In a recent study, XCR1+ cDC1s were increased in number in the blood and liver from patients with NASH, due to enhanced proliferation in the bone marrow. Mechanistically, analysis of DC–T cell pairs in liver-draining lymph nodes demonstrated cDCs potentiate CD8 T cell activation in NASH38. 4.5.3 T Cells During homeostasis, conventional T cells, including CD4+ and, more commonly, CD8+ αβ T cells, perform immune surveillance, along with a subset of liver-resident/memory CD8+ T cells. Infuenced by innate immune cells and the

4 MECHANISMS OF HEPATOCYTE INJURY AND INFLAMMATION IN NAFLD

Figure 4.3

Liver-immune cell crosstalk mediates NASH (This fgure was created with BioRender.com.)

Signals released from lipotoxic hepatocytes in the form of extracellular vesicles (EVs) or damage-associated molecular patterns (DAMPs) recruit various bone marrow-derived immune cells. For example, loss of resident Kupffer cells is followed by reoccupation of the liver macrophage niche by bone marrow-derived monocytes. These cells release proinfammatory cytokines, such as TNFα, IL-1, mediate further hepatocellular injury directly and by crosstalk with other immune cells such as T cells, and activate stellate cells leading to fbrosis. Further, adipose tissue-derived adipokines or gut microbiotaderived products, respectively, lead to activation of intrahepatic proinfammatory macrophage and B cells.

local niche, CD4+ T cell subpopulations include TH1, TH2 and TH17 cells and are characterized by the expression of interferon-γ (IFNγ), IL-4 and/or IL-13, and IL-17, respectively. Considered the prototypical TH1 cytokine, IFNγ can also be produced by other cell types including NK cells, and IFN-γ-defcient mice were protected from NASH39. In human NAFLD, the CXC chemokine receptor 3 (CXCR3), implicated in the chemotaxis of T cells, was signifcantly upregulated in liver biopsies, and CXCR3 knockout mice were protected from NASH40. Cytokines released by TH2 cells have also been studied in NASH; for example, serum levels of IL-13, implicated in HSC activation, are elevated in NASH patients. Intrahepatic TH17 cells were more abundant and activated in human NASH and normalized

after bariatric surgery. Mechanistically, murine models of NASH demonstrate increased glycolytic skewing in TH17 cells and IL-17-induced hepatic lipotoxicity and myeloid infltration41. Finally, in a humanized mouse model of NASH, circulating and intrahepatic CD4+ T cells were correlated with infammation and fbrosis42. In addition to producing infammatory cytokines, CD8+ T cells are directly cytotoxic via effector molecules such as granzymes and perforins. In human NASH, CD8+ accumulate in the liver and promote carcinogenesis by secreting TNF superfamily cytokines; CD8+ T cell depletion blunted murine NASH43. Recently, in both human and murine NASH, a tissue-resident CXCR6+ subset that simultaneously expressed effector (granzyme) and 35

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exhaustion (PD1) markers was described44. Intriguingly, these T cells were “autoaggressive” in that, their activation was antigen independent but susceptible to metabolic stimuli. Consequently, PD1 blockade caused exaggerated CD8+ T cell activation and worsened NASH. The liver also contains innate-like T cell populations, including γδ T cells, natural killer T (NKT) cells, which are abundant in mice, and mucosal-associated invariant T (MAIT) T cells in humans. Microbiota sustain proinfammatory intrahepatic γδ T cells45 and invariant NKT (iNKT) cells in a CD1d-restricted lipid antigen manner. Notably, iNKT cells are variably abundant in mouse and human liver, representing up to 50% and 4 risk alleles)69. Of note, the percentage of genus-level taxa variation explained by our NAFLD/NASH PRS was ~7.4%, which was independent of key covariate parameters that may affect this effect, including age, gender, steatosis score and obesity degree69.To the best of our knowledge, our study is the frst in exploring the interrelationship between the liver metataxonomic profle in patients with NAFLD across the entire spectrum of the disease severity and variants modifying either risk or protection against the disease and variants involved in carbohydrate intake and macronutrient preferences. Together, we have provided evidence that the liver microbiota depends in part on the host genetic background69. These observations may represent potentially actionable mechanisms of disease.

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5.10 CONCLUSION/SUMMARY POINTS Microbiome-derived bioactive molecules are key regulators of virtually every aspect of body physiology. In the context of NAFLD, microbially derived metabolites exert a myriad of modifcations in the liver tissue that are relevant to the NAFLD biology, including activation of pathways associated with liver fat accumulation, infammation and hepatic stellate cell activation. Gut dysbiosis might also drive the development of NAFLD by endogenous alcohol production. The discovery of the liver microbiome in samples of patents with NAFLD across the entire spectrum of the disease severity suggested key fndings to explain the disease biology and probably a portion of the “missing heritability”37. Across major host phenotypic differences—from moderate to severe obesity—the study of the liver metataxonomic prof le identifed distinctive liver microbial DNA patterns associated with key histological features such as disease severity, liver infammation and fbrosis. The strongest severe disease-associated imbalance in bacterial DNA was highly linked to obesity. Whereas overabundance of Proteobacteria (alpha or gamma) was predominantly seen in liver specimens of nonmorbidly obese patients, overrepresentation of Peptostreptococcus-,

5 ROLE OF THE MICROBIOME

Figure 5.5

Conclusion/summary points

NAFLD is a multifactorial disease that denotes phenotypic complexity. The fgure highlights the role of the interrelationship between the host-genome and the liver tissue microbiota, a novel mechanism that may explain the disease biology. There are relevant actors in this relationship, including ethnic diversity and geographic location, the age and gender of the affected patients, and the crucial role of the environmental factors, such as diet.

Verrucomicrobia-, Actinobacteria-, and Proteobacteriaderived DNA was more frequently observed in livers of morbidly obese patients. Notably, decreased amounts of bacterial DNA from the Lachnospiraceae family were associated with more severe histological features. This study suggests that therapeutic options, including probiotic selection, should be precisely defned according to specifc clinical scenarios, including features of the host phenome. Finally, it becomes clear that understanding the putative synergistic effect(s) between the microbiome and their by-products and the genetic background of the host may serve to predict and design novel personalized treatments17. The goal of future investigations in this feld should be identifying novel pathways and interactions with microbial components that could provide effective therapies for NASH. Nevertheless, there are gaps in our understanding of how and by which mechanisms these putative interactions between the body microbiotas and the host-genetics impact on disease biology17. It is obvious that the microbiome research domain is driven by methodological advances. Thus future investigations will accelerate our ability to completely understand the role of the microbiome in the biology of NAFLD. Although progress has been made, unanswered questions remain. Nevertheless, this area of research has a bright future. 5.11 ACKNOWLEDGMENT This study was partially supported by grants PICT 2018– 889, PICT 2019–0528, PICT 2016–0135, PICT2018–00620 (Agencia Nacional de Promoción Científca y Tecnológica, FONCyT), CONICET Proyectos Unidades Ejecutoras 2017, PUE 0055. Conficts of interest: None

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11. Wong VW, Tse CH, Lam TT et al. Molecular characterization of the fecal microbiota in patients with nonalcoholic steatohepatitis-a longitudinal study. PLoS ONE 2013;8(4):e62885. 12. Zhu L, Baker SS, Gill C et al. Characterization of gut microbiomes in nonalcoholic steatohepatitis (NASH) patients: a connection between endogenous alcohol and NASH. Hepatology 2013;57(2):601–609. 13. Behary J, Amorim N, Jiang XT et al. Gut microbiota impact on the peripheral immune response in nonalcoholic fatty liver disease related hepatocellular carcinoma. Nat Commun 2021;12(1):187. 14. Michail S, Lin M, Frey MR et al. Altered gut microbial energy and metabolism in children with non-alcoholic fatty liver disease. FEMS Microbiol Ecol 2015;91(2):1–9. 15. Ponziani FR, Bhoori S, Castelli C et al. Hepatocellular carcinoma is associated with gut microbiota profle and infammation in nonalcoholic fatty liver disease. Hepatology 2019;69(1):107–120. 16. He LH, Yao DH, Wang LY, Zhang L, Bai XL. Gut microbiome-mediated alteration of immunity, infammation, and metabolism involved in the regulation of non-alcoholic fatty liver disease. Front Microbiol 2021;12:761836. 17. Sookoian S, Pirola CJ. Liver tissue microbiota in nonalcoholic liver disease: a change in the paradigm of host-bacterial interactions. Hepatobiliary Surg Nutr 2021;10(3):337–349. 18. Heeg K, Sparwasser T, Lipford GB, Hacker H, Zimmermann S, Wagner H. Bacterial DNA as an evolutionary conserved ligand signalling danger of infection to immune cells. Eur J Clin Microbiol Infect Dis 1998;17(7):464–469. 19. Fujieda S, Iho S, Kimura Y et al. DNA from Mycobacterium bovis bacillus Calmette-Guerin (MY-1) inhibits immunoglobulin E production by human lymphocytes. Am J Respir Crit Care Med 1999;160(6):2056–2061. 20. De VF, Kovatcheva-Datchary P, Goncalves D et al. Microbiota-generated metabolites promote metabolic benefts via gut-brain neural circuits. Cell 2014;156(1–2):84–96. 21. Ren Z, Li A, Jiang J et al. Gut microbiome analysis as a tool towards targeted non-invasive biomarkers for early hepatocellular carcinoma. Gut 2019;68(6):1014–1023. 22. Yoshimoto S, Loo TM, Atarashi K et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature 2013;499(7456):97–101. 23. Demir M, Lang S, Hartmann P et al. The fecal mycobiome in non-alcoholic fatty liver disease. J Hepatol 2021. 24. Hoffmann C, Dollive S, Grunberg S et al. Archaea and fungi of the human gut microbiome: correlations with diet and bacterial residents. PLoS ONE 2013;8(6):e66019. 25. Manrique P, Bolduc B, Walk ST, van der Oost J, de Vos WM, Young MJ. Healthy human gut phageome. Proc Natl Acad Sci U S A 2016;113(37):10400–10405. 26. Hsu CL, Duan Y, Fouts DE, Schnabl B. Intestinal virome and therapeutic potential of bacteriophages in liver disease. J Hepatol 2021;75(6):1465–1475. 27. Manrique P, Zhu Y, van der Oost J et al. Gut bacteriophage dynamics during fecal microbial transplantation in subjects with metabolic syndrome. Gut Microbes 2021;13(1):1–15.

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28. Lang S, Demir M, Martin A et al. Intestinal virome signature associated with severity of nonalcoholic fatty liver disease. Gastroenterology 2020;159(5):1839–1852. 29. Duan Y, Llorente C, Lang S et al. Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease. Nature 2019;575(7783):505–511. 30. Amar J, Lange C, Payros G et al. Blood microbiota dysbiosis is associated with the onset of cardiovascular events in a large general population: the D.E.S.I.R. study. PLoS ONE 2013;8(1):e54461. 31. Koren O, Spor A, Felin J et al. Human oral, gut, and plaque microbiota in patients with atherosclerosis. Proc Natl Acad Sci U S A 2011;108(Suppl 1):4592–4598. 32. Lelouvier B, Servant F, Paisse S et al. Changes in blood microbiota profles associated with liver fbrosis in obese patients: A pilot analysis. Hepatology 2016;64(6):2015–2027. 33. Puri P, Liangpunsakul S, Christensen JE et al. The circulating microbiome signature and inferred functional metagenomics in alcoholic hepatitis. Hepatology 2018;67(4):1284–1302. 34. Cho EJ, Leem S, Kim SA et al. Circulating microbiotabased metagenomic signature for detection of hepatocellular carcinoma. Sci Rep 2019;9(1):7536. 35. Kajihara M, Koido S, Kanai T et al. Characterisation of blood microbiota in patients with liver cirrhosis. Eur J Gastroenterol Hepatol 2019;31(12):1577–1583. 36. Bajaj JS, Betrapally NS, Hylemon PB et al. Salivary microbiota refects changes in gut microbiota in cirrhosis with hepatic encephalopathy. Hepatology 2015;62(4):1260–1271. 37. Sookoian S, Salatino A, Castano GO et al. Intrahepatic bacterial metataxonomic signature in non-alcoholic fatty liver disease. Gut 2020;69(8):1483–1491. 38. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R. Bacterial community variation in human body habitats across space and time. Science 2009;326(5960):1694–1697. 39. Pirola CJ, Sookoian S. The lipidome in nonalcoholic fatty liver disease: actionable targets. J Lipid Res 2021;62:100073. 40. Postler TS, Ghosh S. Understanding the holobiont: How microbial metabolites affect human health and shape the immune system. Cell Metab 2017;26(1):110–130. 41. Willemsen LE, Koetsier MA, van Deventer SJ, van Tol EA. Short chain fatty acids stimulate epithelial mucin 2 expression through differential effects on prostaglandin E(1) and E(2) production by intestinal myofbroblasts. Gut 2003;52(10):1442–1447. 42. Ridlon JM, Kang DJ, Hylemon PB, Bajaj JS. Bile acids and the gut microbiome. Curr Opin Gastroenterol 2014;30(3):332–338. 43. Sookoian S, Flichman D, Castano GO, Pirola CJ. Mendelian randomisation suggests no benefcial effect of moderate alcohol consumption on the severity of nonalcoholic fatty liver disease. Aliment Pharmacol Ther 2016;44(11–12):1224–1234. 44. Vilar-Gomez E, Sookoian S, Pirola CJ et al. ADH1B *2 is associated with reduced severity of nonalcoholic fatty liver disease in adults, independent of alcohol consumption. Gastroenterology 2020;159(3):929–943. 45. Cope K, Risby T, Diehl AM. Increased gastrointestinal ethanol production in obese mice: implications

5 ROLE OF THE MICROBIOME

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for fatty liver disease pathogenesis. Gastroenterology 2000;119(5):1340–1347. Nair S, Cope K, Risby TH, Diehl AM. Obesity and female gender increase breath ethanol concentration: potential implications for the pathogenesis of nonalcoholic steatohepatitis. Am J Gastroenterol 2001;96(4):1200–1204. Yuan J, Chen C, Cui J et al. Fatty liver disease caused by high-alcohol-producing klebsiella pneumoniae. Cell Metab 2019;30(4):675–688. Li NN, Li W, Feng JX et al. High alcohol-producing Klebsiella pneumoniae causes fatty liver disease through 2,3-butanediol fermentation pathway in vivo. Gut Microbes 2021;13(1):1979883. Elshaghabee FM, Bockelmann W, Meske D et al. Ethanol production by selected intestinal microorganisms and lactic acid bacteria growing under different nutritional conditions. Front Microbiol 2016;7:47. Fouts DE, Torralba M, Nelson KE, Brenner DA, Schnabl B. Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease. J Hepatol 2012;56(6):1283–1292. Yan AW, Fouts DE, Brandl J et al. Enteric dysbiosis associated with a mouse model of alcoholic liver disease. Hepatology 2011;53(1):96–105. Henao-Mejia J, Elinav E, Jin C et al. Infammasomemediated dysbiosis regulates progression of NAFLD and obesity. Nature 2012;482(7384):179–185. Guo S, Nighot M, Al-Sadi R, Alhmoud T, Nighot P, Ma TY. Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR4 signal transduction pathway activation of FAK and MyD88. J Immunol 2015;195(10):4999–5010. Goodrich JK, Waters JL, Poole AC et al. Human genetics shape the gut microbiome. Cell 2014;159(4):789–799. Goodrich JK, Davenport ER, Beaumont M et al. Genetic determinants of the gut microbiome in UK twins. Cell Host Microbe 2016;19(5):731–743. Romeo S, Kozlitina J, Xing C et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008;40(12):1461–1465. Sookoian S, Castano GO, Burgueno AL, Gianotti TF, Rosselli MS, Pirola CJ. A nonsynonymous gene variant in the adiponutrin gene is associated with nonalcoholic fatty liver disease severity. J Lipid Res 2009;50(10):2111–2116. Sookoian S, Pirola CJ. Meta-analysis of the infuence of I148M variant of patatin-like phospholipase domain

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containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 2011;53(6):1883–1894. Monga KA, Testerman T, Galuppo B et al. Effect of Gut Microbiota and PNPLA3 rs738409 variant on nonalcoholic fatty liver disease (NAFLD) in obese youth. J Clin Endocrinol Metab 2020;105(10). Kozlitina J, Smagris E, Stender S et al. Exome-wide association study identifes a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2014;46(4):352–356. Milano M, Aghemo A, Mancina RM et al. Transmembrane 6 superfamily member 2 gene E167K variant impacts on steatosis and liver damage in chronic hepatitis C patients. Hepatology 2015;62(1):111–117. Pirola CJ, Sookoian S. The dual and opposite role of the TM6SF2-rs58542926 variant in protecting against cardiovascular disease and conferring risk for nonalcoholic fatty liver: a meta-analysis. Hepatology 2015;62(6):1742–1756. Sookoian S, Castano GO, Scian R et al. Genetic variation in transmembrane 6 superfamily member 2 and the risk of nonalcoholic fatty liver disease and histological disease severity. Hepatology 2015;61(2):515–525. Mancina RM, Dongiovanni P, Petta S et al. The MBOAT7-TMC4 variant rs641738 increases risk of nonalcoholic fatty liver disease in individuals of European descent. Gastroenterology 2016;150(5):1219–1230. Abul-Husn NS, Cheng X, Li AH et al. A protein-truncating HSD17B13 variant and protection from chronic liver disease. N Engl J Med 2018;378(12):1096–1106. Ma Y, Belyaeva OV, Brown PM et al. 17-beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease. Hepatology 2019;69(4):1504–1519. Pirola CJ, Garaycoechea M, Flichman D et al. Splice variant rs72613567 prevents worst histologic outcomes in patients with nonalcoholic fatty liver disease. J Lipid Res 2019;60(1):176–185. Chu AY, Workalemahu T, Paynter NP et al. Novel locus including FGF21 is associated with dietary macronutrient intake. Hum Mol Genet 2013;22(9):1895–1902. Pirola CJ, Salatino A, Quintanilla MF, Castano GO, Garaycoechea M, Sookoian S. The infuence of host genetics on liver microbiome composition in patients with NAFLD. EBioMed 2022. In press.

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SECTION II

DIAGNOSTIC TESTS

53

SECTION II: DIAGNOSTIC TESTS

6 Simple Algorithms in Primary Care Xixi Xu and Michelle T. Long

CONTENTS 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 6.2 Who Is at Risk for NAFLD?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 6.3 Screening for NAFLD: General Population and High-Risk Groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 6.4 Noninvasive Screening Modalities for NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 6.5 What to Do When a Patient Is Suspected of Having NAFLD? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.6 Risk Stratifcation in NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.6.1 Blood-Based Risk Stratifcation Scores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.6.2 Imaging-Based Risk Stratifcation Scores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 6.7 Suggested Risk Stratifcation Algorithm for Primary Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 6.8 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 6.1 INTRODUCTION The global pandemic of obesity impacts an estimated 650 million adults worldwide, and the prevalence is rising1. By 2030, approximately 50% of adults in the US will have obesity, and 25% of adults are expected to develop severe obesity2. Along with the rise in obesity, associated conditions, including nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver disease (NAFLD), will also increase. At present, the prevalence of NAFLD and NASH are high, with an estimated US prevalence of 37% for NAFLD and 14% for NASH3,4. Despite the high prevalence of NAFLD and NASH in the population, the guidance on screening for NAFLD or NASH is unclear. Early identifcation coupled with appropriate clinical intervention may result in improved outcomes and lower mortality for individuals with NAFLD and NASH5. Given the high prevalence of disease, it is prudent that primary care providers are well-versed in the diagnosis, risk stratifcation and management of NAFLD and related cardiometabolic diseases. In this chapter, we present a step-to-step guide for primary care providers to build a knowledge base about identifying and risk-stratifying patients for NAFLD. 6.2 WHO IS AT RISK FOR NAFLD? NAFLD encompasses a constellation of disease phenotypes based on different histological features. The most common and benign NAFLD phenotype is simple hepatic steatosis, which accounts for 75% of those with NAFLD6. However, about 25% of persons with NAFLD have NASH, a progressive NAFLD phenotype associated with hepatic fbrosis, which can lead to end-stage liver disease, hepatocellular carcinoma (HCC) and liver-related death. So who is at risk for NAFLD? Family aggregation studies and twin studies suggest that the development of NAFLD may, in part, be related to genetic susceptibility7,8. Multiple genetic loci associate with NAFLD in the general population, including PNPLA3 (encoding patatinlike phospholipase domaincontaining protein 3) and TM6SF2 (encoding transmembrane 6 superfamily member 2)9. In addition, epigenetic factors, such as DNA methylation remodeling of fbrosis modifer genes, may contribute to gene–environment interactions in the risk of developing fbrosis in NAFLD10,11. From an environmental perspective, an unhealthy lifestyle with low physical activity levels and consuming an unhealthy diet, including diets high in sugar, sodium, and fat and low in fresh 54

fruits and vegetables associate with a high prevalence of NAFLD12–15. Obesity, specifcally abdominal or visceral adiposity, is a major risk factor for NAFLD16. Even a modest weight increase of 2 kg can increase an individual’s risk of developing NAFLD17. Yu et al. (2015) has shown that increased visceral adipose tissue is independently associated with NASH and development of fbrosis17. Type 2 diabetes mellitus (T2DM) is also strongly associated with NAFLD, which has a disease prevalence of over 50% among persons with T2DM with an even higher prevalence for those with poor T2DM control18. Metabolic syndrome is another risk factor associated with NAFLD. In a study with a cohort of 11,647 people, the prevalence of NAFLD was signifcantly higher in individuals with metabolic syndrome, and the prevalence increased as the burden of metabolic risk factors increased19. The presence of metabolic syndrome is also seen in more individuals with advanced hepatic fbrosis19. In addition, the risk of developing cirrhosis or hepatocellular carcinoma (HCC) also increases with each additional metabolic syndrome factor. For example, in patients with NAFLD, having both hypertension and dyslipidemia would result in 1.8-fold increase in the risk of developing cirrhosis or HCC20. Though strongly associated with obesity, NAFLD can also exist in persons with normal weight BMI. Lean NAFLD was frst described in Asian populations; however, subsequent studies have observed that between 10 and 20% of Americans and Europeans with NAFLD are lean 21,22. Lean individuals with NAFLD often have a higher burden of cardiometabolic disease risk factors compared to lean individuals without NAFLD23. Additionally, the risk of cardiovascular disease events may be similar or higher for lean persons with NAFLD compared to overweight and obese persons with NAFLD24,25. Having normal weight does not reduce the risk of fbrosis progression or mortality from NAFLD26. 6.3 SCREENING FOR NAFLD: GENERAL POPULATION AND HIGH-RISK GROUPS As the presence of NAFLD in the population increases, the morbidity and mortality due to disease progression and complication, including cirrhosis, liver failure and HCC, will rapidly rise. At present, NAFLD is the second most common etiology of chronic liver disease among patients listed DOI: 10.1201/9781003386698-8

6 SIMPLE ALGORITHMS IN PRIMARY CARE

for liver transplant in the United States27. The prevalence of NAFLD as an indication for liver transplant has almost doubled between 2004 and 201328. The cost associated with diagnosing and treating NAFLD is expected to take up a signifcant portion of total health care spending. In 2016, the annual economic burden of NAFLD in the US was estimated to be $103 billion29, and costs are expected to increase with the rising prevalence. Therefore, early detection and early intervention for patients who are at risk for progressive NAFLD are critical to prevent morbidity and mortality. Whereas it is reasonable to assume that routine screening may improve early detection and intervention, there is no consensus on routine screening for NAFLD. The US Preventive Services Task Force (USPSTF) and National Institute for Health and Care Excellence (NICE) make no recommendations for or against general screening for NAFLD. The American Association for the Study of Liver Diseases (AASLD) practice guidelines do not recommend routine screening for NAFLD, citing the lack of evidence on cost-effectiveness and effective treatments30, though data on cost-effectiveness are now available since the publication of the guidelines. The European Association for the Study of the Liver (EASL), on the contrary, has recommended screening for every person with persistently abnormal liver biochemical tests31. With the emerging of more effective medications, screening for NAFLD among high-risk patient populations is likely to be cost-effective. It is well-established that patients with T2DM and metabolic syndrome are at increased risk of developing NAFLD32. Particularly, T2DM is a signifcant risk factor for the development of NAFLD, liver fbrosis and HCC33–35. The AASLD guidelines recommend that clinicians should have a high index of suspicion for NAFLD and NASH in patients with T2DM and encourage the use of clinical tools, such as the NAFLD Fibrosis Score (NFS), the Fibrosis-4 index (FIB-4) or vibrationcontrolled transient elastography, to further risk-stratify30. The American Diabetes Association (ADA) also recognizes the close association of T2DM with the development of NAFLD; thus the 2020 ADA guidelines propose to screen all patients with T2DM, prediabetes, elevated liver aminotransferase levels or fatty liver on ultrasound for NAFLD36. Noureddin et al. (2020) have demonstrated that screening for NAFLD in patients with T2DM with ultrasound and liver biochemical tests, followed by noninvasive testing for fbrosis is cost-effective37. Although further research is necessary to uncover additional risk factors for NAFLD, it is reasonable to identify patients with T2DM, multiple metabolic risk factors, abnormal liver aminotransferases, or incidental hepatic steatosis on imaging in the primary care setting as patients at high risk for NAFLD. Thus American Gastroenterology Association (AGA) published an NAFLD/NASH Clinical Care Pathway for this purpose through a multidisciplinary task force with the ADA, American Osteopathic Association, Endocrine Society and the Obesity Society3. 6.4 NONINVASIVE SCREENING MODALITIES FOR NAFLD Liver biopsy, the gold standard for diagnosis NAFLD, is not an appropriate screening test for NAFLD given the highly invasive nature of the test, risk for complications, and high cost. An ideal screening test should be inexpensive, easy to perform, and have relatively high sensitivity. Given the high prevalence of NAFLD in patients with T2DM,

metabolic risk factors, elevated aminotransferases and incidental fndings of steatosis as mentioned in the previous section, the best screening method for primary care providers may be a high clinical suspicion for NAFLD in patients who are at risk. The very initial screening strategy should include detailed history, comprehensive physical exam, focused laboratory tests and abdominal ultrasound. For patients who have risk factors for NAFLD, a detailed history and full physical examination are obtained. Specifcally, clinicians should assess the patients’ alcohol consumption, drug use, sexual history, medication history, herbal or alternative medication use, occupational exposure, BMI, past medical history (particularly history of diabetes, hyperlipidemia and hypertension) and family history of liver diseases such as Wilson’s disease, hemochromatosis, autoimmune disease and alpha-1 antitrypsin defciency38. If elevated liver biochemical tests are detected, clinicians should consider other causes of liver disease as possible contributing factors (Table 6.1). Depending on the pattern of liver biochemical test elevation, liver diseases that should be considered include viral hepatitis, autoimmune hepatitis, Wilson disease, hemochromatosis, alpha-1 antitrypsin defciency, drug-induced liver injury and alcohol-related liver disease. The most common bloodbased screening test for NAFLD are liver biochemical tests, specifcally the serum aminotransferases (alanine aminotransferase [ALT]) and aspartate aminotransferase [AST]). Elevations of ALT or AST may be an indicator of NAFLD with a usual pattern of ALT greater than AST. Along with this pattern of transaminases elevation, γ glutamyltransferase (GGT) may also be elevated39. Many patients with NAFLD or NASH are found to have abnormal ALT40. Thus patients with incidental f ndings of abnormal liver biochemical tests warrant further workup. However, AST and ALT can be normal in up to 78% of patients with NAFLD22; thus serum aminotansferases may not be sensitive for NAFLD. It is important to keep in mind that normal liver biochemical tests do not rule out possible underlying NAFLD in an asymptomatic patient with risk factors. For patients with chronically elevated liver biochemical tests as well as metabolic risk factors such as obesity, diabetes, hyperlipidemia, and hypertension, the most common cause to consider is NAFLD. Contrary to the nomenclature often used, ALT, AST, ALP, GGT and bilirubin are markers of liver injury rather than liver function. The markers for hepatocellular function are albumin, bilirubin and prothrombin time. In order to identify abnormal liver biochemical tests, true healthy normal levels are needed. Establishing a normal range for AST and ALT has been problematic given that reference standards were established in a general population, many of whom likely had undiagnosed NAFLD. In the past, abnormal ALT levels were based on multiples of upper limits of normal to avoid specifcally defning the normal level of ALT41. This method often runs into the issue of different defnitions of upper limits of normal by laboratories, making comparison of liver blood tests from different laboratories challenging. To defne a unifed lower threshold for ALT, patient factors, such as age, sex, medication and supplement use, alcohol use, BMI, and underlying conditions, such as NAFLD and viral hepatitis status, should be taken into consideration when selecting healthy reference populations. The American College of Gastroenterology currently recommends a true healthy normal ALT level to be 29–33 IU/l for men and 19–25 IU/l for women. However, a recent 55

56

Table 6.1: Patterns of Abnormal Liver Function Tests for Diseases Pattern Type Hepatocellular ALT ( & AST)

Causes

Total Bilirubin

Borderline -4× ULN

ALT

Borderline -4× ULN

AST

Normal

Normal

Alcohol-related liver disease Viral infections

Borderline—4× ULN

Borderline—4× ULN

Normal

Normal

Borderline—5× ULN (acute infection >1,000)

Borderline—5× ULN (acute infection >1,000)

Normal

Normal

Congestive hepatopathy Autoimmune hepatitis

Borderline—3× ULN Borderline—5× ULN (acute presentation >10–20× ULN) Borderline -5× ULN

Normal—mildly elevated ALK: AST (or ALT) 50× ULN

Elevated >50× ULN

Normal

Medication and toxins Biliary obstruction Intrahepatic cholestasis Biliary epithelial damage

Variable Normal—mildly elevated

Variable Normal—mildly elevated

Normal ≥ 4× ULN

ALK: Tbili 2 Check HBsAg, HBsAb, HBcAb, HCV Ab and PCR, HAV IgG/IgM, HSV, EBV, and CMV Elevated indirect bilirubin ANA, AMA, ASMA, LKM-1, IgG  Serum Ceruloplasmin and  urine copper Abdominal ultrasound with doppler GGT to confrm hepatic origin, Abdominal ultrasound, MRCP/ERCP if has obstruction. ANA, AMA, ASMA if no obstruction

ANA: Antinuclear antibodies; ASMA: Anti-smooth muscle antibodies; AMA: Anti-mitochondrial antibodies; LKM-1: Liver kidney microsome type 1; MRCP: Magnetic resonance cholangiopancreatography; ERCP: Endoscopic retrograde cholangiopancreatography; GGT: gamma-glutamyl transferase.

SECTION II: DIAGNOSTIC TESTS

Cholesteric ALK ( and direct bilirubin)

Alkaline Phosphatase

NAFLD

6 SIMPLE ALGORITHMS IN PRIMARY CARE

study suggests that age-specifc cutoffs may be more appropriate as 10% of healthy men under the age of 45 years had an elevated ALT42. Clinical judgement remains to be the foremost important factor when interpretations liver biochemical tests of an individual patient. The most common imaging-based screening test for NAFLD is abdominal ultrasound (US). US is an easily accessible, noninvasive way to identify hepatic steatosis qualitatively. The criteria to assess hepatic steatosis via US include hepatorenal echo contrast, liver brightness, deep attenuation and vascular blurring38. The sensitivity for US to detect small amounts of hepatic steatosis is limited; the sensitivity for detection of steatosis is 64% in the general population43, though in higher-risk groups with a higher prevalence of steatosis, the sensitivity of US for steatosis improves to 90%43,44. The specifcity of US in detecting hepatic steatosis remains excellent at >95% regardless of the degree of hepatic steatosis43. However, it is important to clarify that hepatic steatosis does not directly translate to a diagnosis of NAFLD as many other liver or systemic diseases can also show hepatic steatosis on US (Table 6.2). 6.5 WHAT TO DO WHEN A PATIENT IS SUSPECTED OF HAVING NAFLD? To help primary care providers decide on the urgency of management and referral when patients are suspected of having NAFLD, it is critical to review the natural history of NAFLD. In a retrospective study that followed individuals for up to 10 years, the annual incidence of compensated cirrhosis, decompensated cirrhosis and death in patients with NAFLD were 0.28, 0.31 and 0.63%, respectively45. A recent prospective study that followed patients with NAFLD for a period of approximately 4 years showed similar fndings of slow disease progression, low incidence of decompensated cirrhosis and low incidence of HCC in NASH with cirrhosis46. Overall, these studies suggested that NAFLD is a slowly progressive disease, and most people with NAFLD do not have NASH or fbrosis. Individuals with NASH, particularly those with associated hepatic fbrosis, are at increased risk of liver-related events and worsened overall mortality46,47. Primary care providers play a frontline role in the identifcation and risk stratifcation of patients with NAFLD in order to identify those with hepatic fbrosis and most in need of liver-directed care.

6.6 RISK STRATIFICATION IN NAFLD 6.6.1 Blood-Based Risk Stratifcation Scores Some of the commonly used serology tests to assess the risk of disease progression to NASH and advanced fbrosis are FIB-4, NAFLD fbrosis score (NFS) and enhanced liver fbrosis (ELF) (Table 6.3). Originally used to predict level of fbrosis for patients with Hepatitis C, FIB-4 index is a simple scoring system that combines patient age with AST, ALT and platelet count48. FIB-4 was subsequently validated to predict risk for advanced fbrosis in NAFLD for both obese and nonobese patient populations49,50. As one of the earliest and most well-studied noninvasive tests for detecting fbrosis, FIB-4 is endorsed by the American College of Gastroenterology as a risk stratifcation tool in clinical practice51. A FIB-4 value less than 1.3, summarized from various studies, showed >90% of negative predictive value to rule out advanced fbrosis52. Greater than 90% of positive predictive value for detecting cirrhosis is achieved with a FIB-4 value > 2.6853. It is important to note that FIB-4 is unable to accurately diagnose advance fbrosis in patients who are younger than 35 or older than 65 years of age53. Patients who are older than 65 years old may require a different cutoff (FIB-4 > 2.0) from the aforementioned values53. NFS is another scoring system that is widely validated for predicting advanced fbrosis from NAFLD. NFS includes patient characteristics such as age, BMI, fasting blood glucose, AST, ALT, albumin and platelet count. With a threshold of −1.455, the NFS has a negative predictive value of about 90%, a sensitivity of 72% and a specifcity of 73%, which suggests that this test can be useful to rule out advanced fbrosis in persons with NAFLD. NFS has modest to high accuracy to diagnose advance fbrosis with a cutoff value of 0.676 with positive predictive value of around 80%52,54. ELF was validated in 2017 as a serology-based scoring system to detect liver fbrosis and NASH. Different from FIB-4 and NFS, the ELF score is based on specialized tests that may not be commonly utilized in the primary care setting. The scoring test is based on the level of hyaluronic acid, procollagen III amino-terminal peptide and tissue inhibitor of matrix metalloproteinase I. For the cutoff value 9.0, the ELF test has a sensitivity of 85.7%, a specifcity of 83.3%,

Table 6.2: Common Differential Diagnosis of Hepatic Steatosis on Imaging Liver-Specifc Disease • NAFLD • Alcoholic fatty liver disease • Acute fatty liver of pregnancy • Chronic hepatitis C (especially genotype 3) • HIV • Wilson’s disease • Hemochromatosis • α1- antitrypsin defciency

Endocrine • Hypothyroidism • Hypopituitarism • Polycystic ovary syndrome • Growth hormone insuffciency

Inborn Errors of Metabolism • Lysosomal acid lipase defciency (LAL-D) • Familial hypobetalipoprotein B • Abetalipoproteinemia • Urea cycle disorders • Hereditary fructose intolerance • Glycogen storage disease • Fatty acid oxidation disorders • Autosomal recessive carbamoylphosphate synthetase I (CPSI) defciency

Acquired Metabolic Disorder • Infammatory bowel disease • Celiac disease • Jejunoileal bypass • Malnutrition/ Kwashiorkor disease • Acute weight loss • Short bowel syndrome • Total parenteral nutrition

Drug and Toxin • • • • • • •

Methotrexate Amiodarone Corticosteroids Valproic acid Tetracycline Amphetamines Older generation of NRTI: didanosine, stavudine and zidovudine • Heavy metal: lead, cadmium and mercury • Herbicide and pesticide

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Table 6.3: Comparison of Different Noninvasive Tests for Detecting Advanced Fibrosis in NAFLD PPV (%) NPV (%) Advantages and Disadvantages

Cutoff

Sensitivity (%)

Specifcity (%)

ALT

< 35 > 70

68.9 40

22.6 57.6

AST/ALT ratio2

< 0.8 > 1.0 1

74 52 27

78 90 89

44 55 37

93 89 84

< 1.3 > 2.67

85 33

65 98

36 80

95 83

NFS2

< −1.455 > 0.676

78 33

58 98

30 76

92 86

VCTE4,5

< 7.9 kPa > 12.1 kPa

77.3 52

68.8 90

44 71

90.5 80

MRE6

< 3.02 kPa > 3.64 kPa

55.4 86.4

90.7 90.5

91.1 67.9

54.2 96.6

Test 1

AST to platelet ratio (APRI)2 FIB-4 index2,3

Easily accessible test, and most patients would have ALT measured from annual physical checkup at some point. Poor sensitivity and specifcity to detect NASH and advance fbrosis Both AST and ALT can be easily obtained. Positive predictive value remains low and will miss patients with advance fbrosis Easily calculated from platelet count, patient’s age, AST and ALT. FIB-4 index has been well-validated as a noninvasive test for advanced fbrosis in NAFLD. The test is limited to patients who are between the ages of 35 and 65 More complex calculation from patient’s age, BMI, presence of diabetes, AST, ALT, platelets and albumin. NFS has been well-validated as a noninvasive test for advance fbrosis in NAFLD. Relatively easy and fast test that can be performed in offce with lower cost. The test accuracy is limited by patient’s BMI, and results may be invalid in patients with BMI > 35. Better accuracy compared to VCTE and provides comprehensive evaluation of the whole liver. Very limited availability given high cost. Requires specialized technician and radiologist to conduct and interpret the study.

PPV: Positive predictive value; NPV: Negative predictive value; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; NFS: NAFLD fbrosis score; BMI: body mass index. 1.

2.

3.

4.

5.

6.

Verma S, Jensen D, Hart J, Mohanty SR. Predictive value of ALT levels for non-alcoholic steatohepatitis (NASH) and advanced fbrosis in non-alcoholic fatty liver disease (NAFLD). Liver Int. Oct 2013;33(9):1398–1405. doi:10.1111/liv.12226 McPherson S, Stewart SF, Henderson E, Burt AD, Day CP. Simple non-invasive fbrosis scoring systems can reliably exclude advanced fbrosis in patients with non-alcoholic fatty liver disease. Gut. Sep 2010;59(9):1265–1269. doi:10.1136/gut.2010.216077 Rikhi R, Singh T, Modaresi Esfeh J. Work up of fatty liver by primary care physicians, review. Ann Med Surg (Lond). Feb 2020;50:41–48. doi:10.1016/j.amsu.2020.01.001 Wong VW, Vergniol J, Wong GL, et al. Diagnosis of fbrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease. Hepatology. Feb 2010;51(2):454–462. doi:10.1002/hep.23312 Siddiqui MS, Yamada G, Vuppalanchi R, et al. Diagnostic accuracy of noninvasive fbrosis models to detect change in fbrosis stage. Clin Gastroenterol Hepatol. Aug 2019;17(9):1877–1885.e5. doi:10.1016/j.cgh.2018.12.031 Loomba R, Wolfson T, Ang B, et al. Magnetic resonance elastography predicts advanced fbrosis in patients with nonalcoholic fatty liver disease: a prospective study. Hepatology. Dec 2014;60(6):1920–1928. doi:10.1002/hep.27362

a positive predictive value of 60.0% and negative predictive value of 95.2%. This test can reliably differentiate mild fbrosis (F0/F1) from advanced fbrosis (F2/F3) in patients with NAFLD55. A high ELF score (>11.3) may predict a higher risk of developing liver-related outcomes at 1 year, better than FIB-4 or the Model for End-Stage Liver Disease (MELD) score56. The ELF test was approved by the FDA in 2021; however, the specifc cutoff value that applies to the real-world clinical practice in the US remains to be determined. However, these noninvasive serology tests can be limited as they might not be applicable to certain populations. For example, FIB-4 has been shown to be inaccurate for older individuals (>65 years old) or those with chronic kidney diseases53,57. In addition, NFS and FIB-4 both use lower and 58

upper thresholds to maximize sensitivity or specifcity when ruling in or ruling out advanced fbrosis. However, many individuals may fall between the cutoffs into an indeterminate zone, which may still require additional testing to rule out advanced disease. Therefore, a second test may be necessary to further risk-stratify the patients in the indeterminate group. 6.6.2 Imaging-Based Risk Stratifcation Scores Supplementing the serology tests, several imaging modalities are validated to assess liver stiffness, a surrogate measure of liver fbrosis. Common imaging modalities include vibration-controlled transient elastography (VCTE), magnetic resonance elastography (MRE) and ultrasoundbased 2D shear wave elastrography (2D-SWE).

6 SIMPLE ALGORITHMS IN PRIMARY CARE

VCTE measures liver stiffness by measuring shear wave velocity of a low-frequency elastic wave directed into the liver from an ultrasound probe. It is a relatively simple and fast test that can be performed in the offce. Liver stiffness measurements have been shown to correlate with the degree of liver fbrosis in NAFLD58,59. Wong et al. (2010) demonstrated that the area under the receiver-operating characteristic curve of VCTE is signifcantly better compared to that of serology-based test for liver fbrosis such as FIB-4, NFS and AST-to-platelet ratio index. For a cutoff value of 7.9 kPa, the negative predictive value for advanced fbrosis is 96%; however, the positive predictive value is only at 52%. With liver stiffness measurement less than 7.9 kPa, the VCTE is best used to rule out advanced fbrosis59. Other studies have shown that for a cutoff value of 8.2 kPa for fbrosis stage ≥ 2, the negative predictive value is 97% in the general population and 78% among those with T2DM60. An upper cutoff threshold of 15 kPa has a positive predictive value and negative predictive value for advanced fbrosis of 77.8 and 100%, respectively61. Based on the actual prevalence of NAFLD in the population, with the top threshold of 12.1 kPa, the positive predictive value to include patients with fbrosis stage ≥ 2 is 88%. Individuals with a liver stiffness >12.1 kPa on VCTE are likely to have some degree of hepatic fbrosis60. Magnetic resonance elastography (MRE) is another clinically useful tool for evaluating the stage of fbrosis. Similar to VCTE, MRE utilizes MRI to propagate acoustic shear waves into the liver, and the liver stiffness measurements can be calculated based on the propagation of the shear waves. For patients with liver stiffness measurements greater than 3.63 kPa, MRE is an accurate test to diagnose advance fbrosis with the sensitivity, specifcity, positive predictive value and negative predictive value of 86, 91, 68 and 97%, respectively62. MRE is a superior test compared to VCTE not only because of better accuracy, but also MRE provides a comprehensive evaluation of the whole liver instead of the limited area evaluated by VCTE63. For patients with severe abdominal obesity, MRE is also a better test compared to other noninvasive imaging modalities for liver fbrosis64. At this time, MRE is not widely used as a point-of-care imaging test due to its high cost and requirement for specialists to interpret the images. Ultrasound-based 2D-SWE is a novel imaging modality that utilizes a focused ultrasound beam to pass shear waves through an area of liver tissue of interest. 2D-SWE is performed using a conventional ultrasound probe, so it shares the same beneft of ultrasound to obtain real-time and quantitative measures of different areas of the liver. 2D-SWE has been shown to have comparably good diagnostic accuracy for advanced fbrosis as MRE and VCTE, but it is more accessible and easier to operate compared to MRE65. 6.7 SUGGESTED RISK STRATIFICATION ALGORITHM FOR PRIMARY CARE Studies have shown that noninvasive tests such as serology and imaging tests are adequate to risk-stratify patients in the primary care setting and to monitor for NAFLD progression. Utilizing two-step algorithms, unnecessary referrals can be reduced by up to 80% while increasing detection of advanced fbrosis and cirrhosis by approximately 5 times66,67. Using a simulation model of American patients with NAFLD, Tapper et al. (2016) showed that using NFS alone or the combination of NFS and VCTE in the primary care clinic is the most cost-effective strategy to risk-stratify NAFLD 68. Other studies based on the US and

European populations have also confrmed that the combination of FIB-4 or NFS with VCTE has diagnostic accuracy and is cost-effective69–71. Based the multiple proposed algorithms in the literatures, we proposed an algorithm for primary care providers in identifying, screening and fbrosis risk-assessing patients for NAFLD3,72,73 (Figure 6.1). The algorithms to use in the primary care offce that are currently published in major guidelines generally follow a two-step model. Clinical practice guideline from the European Association for the Study of the Liver (EASL), European Association for the Study of Diabetes (EASD) and European Association for the Study of Obesity (EASO) suggested a diagnostic fowchart to assess and monitor disease severity in patients with suspected NAFLD or metabolic risk factors. The frst diagnostic step recommended by this guideline is liver blood tests (ALT, AST and GGT) and ultrasound. When imaging modalities are not available, then serum biomarkers and scores are acceptable alternatives for diagnosing NAFLD72. If there are signs of fatty liver on ultrasound and normal liver blood tests, the second step of the algorithm is to check serum fbrosis markers such as NFS, FIB-4 or ELF. Based on the risk suggested by the serum fbrosis marker, patients with low risk should have a follow-up in 2 years with repeated liver blood tests and serum fbrosis markers. For patients with medium/high risk based on serum fbrosis markers, referral to Hepatology is necessary for a more thorough assessment of disease severity and evaluation for liver biopsy. Similarly, patients with abnormal liver blood tests and signs of fatty liver disease on ultrasound will also require evaluation from specialists. For patients with normal liver ultrasound and liver blood tests, repeated ultrasound and liver blood tests should be done in 3–5 years72. Multiple publications have also suggested similar algorithms to screen patients who are at risk for NAFLD. The frst step is to identify patients who at risk of NAFLD, which is defned as patient with more than 2 metabolic risk factors (central obesity, hyperlipidemia, hypertension, prediabetes or insulin resistance), type 2 diabetes, signs of fatty liver on any imaging modalities, or elevated aminotransferases. Once patients at risk are identifed, step 2 is to rule out other pathology that can lead to liver diseases. Extensive past medical history, social history, medication history and physical exams are necessary for all patients with suspected NAFLD3,73–76. The next step is risk stratifcation for the risk of hepatic fbrosis in patients who are at risk of NAFLD with noninvasive testing such as FIB-4, NFS, or ELF. Most of the established algorithms utilize FIB-4 and NFS. ELF is a relatively new serum score test that requires specialized serum tests, and it is mostly utilized in European guidelines. ELF was recently approved in the US to monitor fbrosis in chronic liver disease but not to screen for hepatic fbrosis. More research on ELF is needed before it can be recommended as a tool in primary clinic to evaluate for fbrosis. FIB-4 and NFS have been shown to have superior diagnostic accuracy to differentiate no fbrosis (F0 and F1) from advanced fbrosis (F3 and F4) compared to other noninvasive fbrosis scores77,78. FIB-4 may also be slightly more accurate than NFS49,79. In addition, the combinations of noninvasive fbrosis scores such as FIB-4 and aspartate transaminase-to-platelet ratio index or FIB-4 and ELF can also be used together to further increase the diagnostic accuracy77,80. Nevertheless, moderate fbrosis is diffcult to 59

SECTION II: DIAGNOSTIC TESTS

Figure 6.1

60

Algorithm for identifying, screening and fbrosis risk-assessing patients for NAFLD

6 SIMPLE ALGORITHMS IN PRIMARY CARE

determine with any accuracy, and it may not be correctly differentiated by noninvasive fbrosis tests. Multiple studies have shown that setting a lower threshold of 1.3 for FIB-4 has a negative protective value of greater than 90%, which could reliably rule out advanced fbrosis50,53,77,78. Thus, for patients who have FIB-4 score 2.67, they are very likely to have advanced fbrosis and require evaluation by a hepatologist and possibly a liver biopsy3. Nevertheless, it should be emphasized that the FIB-4 index is validated in patients aged between 35 and 65 years of age. Different cutoff values, such as FIB-4 >2 as suggested by some studies, may be necessary for patients who are older than 65 years of age53. The remaining group of patients who fall into the intermediate risk group with FIB-4 score between 1.3 and 2.67 make up to 40% of patients with suspected NAFLD35. The intermediate risk group requires secondary evaluation with liver stiffness measurement to differentiate the risk of fbrosis in this group. Another study has suggested a combination of NFS and FIB-4 score as the initial step to categorize patients’ risk of developing advanced fbrosis. NFS by itself already has good diagnostic accuracy for advanced fbrosis. NFS less than −1.455 can safely exclude patients with advanced fbrosis with a negative predictive value >90%. Using an NFS greater than 0.676, the positive predictive value is around 80–90% which can adequately rule in patients with advanced fbrosis52,54. Pandyarajan et al. (2019) proposed a model to combine both NFS and FIB-4 scores together for the initial step of risk stratifcation. For patients with NFS 0.676 and FIB-4 >2.67, the risk of advanced fbrosis is likely to be very high, and the patient would beneft from further evaluation and management by Hepatology. The intermediate group refers to patients who have NFS between −1.455 and 0.676 or FIB-4 between 1.30 and 2.67. Patients in the intermediate group should undergo further risk stratifcation with imaging, such as VCTE for the measurement of the liver stiffness, in order to elucidate the risk of advanced fbrosis73. VCTE, MRE and 2D-SWE are all imaging modalities that can be used to measure liver stiffness. Patients with liver stiffness measurement less than 8.0 kPa on VCTE are less likely to have signifcant hepatic fbrosis59,60, and this low-fbrosis-risk population can be managed by primary care providers with referral to nutrition, counseling on diet, weight loss and repeated noninvasive testing in 2–3 years3. With a liver stiffness measurement >12.0 kPa, the risk of liver fbrosis is very high60,82. The high-risk population warrants referral to Hepatology for further evaluation with liver biopsy or MRE. With further evaluation by Hepatology, patients with high risk may be appropriate for medication

and secondary prevention strategies3. In addition, it is also helpful for primary care providers to be aware that a high liver stiffness measure of >20 kPa suggests high risk of cirrhosis with esophageal varices83. Therefore, patients with a FIB-4 score of > 2.67 and a liver stiffness measure of >20kPa should be promptly referred to Hepatology. Unfortunately, a proportion of patients still fall into the intermediate range of liver stiffness measurement (i.e., 8.0–12.0 kPa). There is no clear guideline on additional testing or management. The decision largely relies on shared decision making between the patient and the primary care provider or specialist. Given that the patient is at risk for NAFLD and has two indeterminate noninvasive testing results, it is reasonable to refer the patient to Hepatology for MRE or liver biopsy for further evaluation. However, if the patient prefers to wait, individual preferences should also be taken into consideration. Primary care providers can discuss with patient about close follow-up and repeating measurement of liver stiffness in 1 year3. 6.8 SUMMARY An increasing evidence base supports the cost-effectiveness of screening for NAFLD, particularly among high-risk groups and as new medications and treatments evolve. Primary care providers play a critical role in the identifcation and initial risk stratifcation of persons at risk for NAFLD. With success, earlier detection and risk stratifcation will prompt early interventions to prevent advanced fbrosis and improve the lives of patients living with NAFLD. REFERENCES 1. The Lancet Gastroenterology H. Obesity: another ongoing pandemic. Lancet Gastroenterol Hepatol. 2021;6(6):411. doi:10.1016/s2468-1253(21)00143-6 2. Ward ZJ, Bleich SN, Cradock AL, et al. Projected U.S. state-level prevalence of adult obesity and severe obesity. N Engl J Med. 2019;381(25):2440–2450. doi:10.1056/ NEJMsa1909301 3. Kanwal F, Shubrook JH, Adams LA, et al. Clinical care pathway for the risk stratifcation and management of patients with nonalcoholic fatty liver disease. Gastroenterology. 2021;161(5):1657–1669. doi:10.1053/j. gastro.2021.07.049 4. Harrison SA, Gawrieh S, Roberts K, et al. Prospective evaluation of the prevalence of non-alcoholic fatty liver disease and steatohepatitis in a large middle-aged US cohort. J Hepatol. 2021;75(2):284–291. doi:10.1016/j. jhep.2021.02.034 5. Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, et al. Weight loss through lifestyle modifcation signifcantly reduces features of nonalcoholic steatohepatitis. Gastroenterology. 2015;149(2):367–378.e5; quiz e14–15. doi:10.1053/j.gastro.2015.04.005 6. Rinella ME. Nonalcoholic fatty liver disease: a systematic review. Jama. 9 2015;313(22):2263–2273. doi:10.1001/ jama.2015.5370 7. Schwimmer JB, Celedon MA, Lavine JE, et al. Heritability of nonalcoholic fatty liver disease. Gastroenterology. 2009;136(5):1585–1592. doi:10.1053/j. gastro.2009.01.050 8. Loomba R, Schork N, Chen CH, et al. Heritability of hepatic fbrosis and steatosis based on a prospective twin study. Gastroenterology. 2015;149(7):1784–1793. doi:10.1053/j.gastro.2015.08.011

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analysis. Gastroenterology. 2020;159(5):1985–1987.e4. doi:10.1053/j.gastro.2020.07.050 Torres DM, Harrison SA. Diagnosis and therapy of nonalcoholic steatohepatitis. Gastroenterology. 2008;134(6):1682–1698. doi:10.1053/j. gastro.2008.02.077 Sattar N, Forrest E, Preiss D. Non-alcoholic fatty liver disease. BMJ. 2014;349:g4596. doi:10.1136/bmj.g4596 Ma X, Liu S, Zhang J, et al. Proportion of NAFLD patients with normal ALT value in overall NAFLD patients: a systematic review and meta-analysis. BMC Gastroenterology. 2020;20(1):1–8. Green RM, Flamm S. AGA technical review on the evaluation of liver chemistry tests. Gastroenterology. 2002;123(4):1367–1384. doi:10.1053/gast.2002.36061 Petroff D, Bätz O, Jedrysiak K, Kramer J, Berg T, Wiegand J. Age dependence of liver enzymes: an analysis of over 1,300,000 consecutive blood samples. Clin Gastroenterol Hepatol. 2022;20(3):641–650. doi:10.1016/j.cgh.2021.01.039 Palmentieri B, de Sio I, La Mura V, et al. The role of bright liver echo pattern on ultrasound B-mode examination in the diagnosis of liver steatosis. Dig Liver Dis. 2006;38(7):485–489. doi:10.1016/j. dld.2006.03.021 Saadeh S, Younossi ZM, Remer EM, et al. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology. 2002;123(3):745–750. doi:10.1053/ gast.2002.35354 Nyberg LM, Cheetham TC, Patton HM, et al. The natural history of NAFLD, a community-based study at a large health care delivery system in the United States. Hepatol Commun. 2021;5(1):83–96. doi:10.1002/hep4.1625 Sanyal AJ, Van Natta ML, Clark J, et al. Prospective study of outcomes in adults with nonalcoholic fatty liver disease. N Engl J Med. 2021;385(17):1559–1569. doi:10.1056/NEJMoa2029349 Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver fbrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology. 2015;149(2):389–397.e10. doi:10.1053/j.gastro.2015.04.043 Sterling RK, Lissen E, Clumeck N, et al. Development of a simple noninvasive index to predict signifcant fbrosis in patients with HIV/HCV coinfection. Hepatology. 2006;43(6):1317–1325. doi:10.1002/hep.21178 Drolz A, Wolter S, Wehmeyer MH, et al. Performance of non-invasive fbrosis scores in non-alcoholic fatty liver disease with and without morbid obesity. Int J Obes (Lond). 2021;45(10):2197–2204. doi:10.1038/ s41366-021-00881-8 Shah AG, Lydecker A, Murray K, Tetri BN, Contos MJ, Sanyal AJ. Comparison of noninvasive markers of fbrosis in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2009;7(10):1104–1112. doi:10.1016/j.cgh.2009.05.033 Younossi ZM, Noureddin M, Bernstein D, et al. Role of noninvasive tests in clinical gastroenterology practices to identify patients with nonalcoholic steatohepatitis at high risk of adverse outcomes: expert panel recommendations. Am J Gastroenterol. 2021;116(2):254–262. doi:10.14309/ajg.0000000000001054 Xiao G, Zhu S, Xiao X, Yan L, Yang J, Wu G. Comparison of laboratory tests, ultrasound, or magnetic resonance elastography to detect fbrosis in

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patients with nonalcoholic fatty liver disease: a metaanalysis. Hepatology. 2017;66(5):1486–1501. doi:10.1002/ hep.29302 McPherson S, Hardy T, Dufour JF, et al. Age as a confounding factor for the accurate non-invasive diagnosis of advanced NAFLD fibrosis. Am J Gastroenterol. 2017;112(5):740–751. doi:10.1038/ ajg.2016.453 Angulo P, Hui JM, Marchesini G, et al. The NAFLD fbrosis score: a noninvasive system that identifes liver fbrosis in patients with NAFLD. Hepatology. 2007;45(4):846–854. doi:10.1002/hep.21496 Polyzos SA, Slavakis A, Koumerkeridis G, Katsinelos P, Kountouras J. Noninvasive liver fbrosis tests in patients with nonalcoholic fatty liver disease: an external validation cohort. Horm Metab Res. 2019;51(2):134–140. doi:10.1055/a-0713-1330 Are VS, Vuppalanchi R, Vilar-Gomez E, Chalasani N. Enhanced liver fbrosis score can be used to predict liver-related events in patients with nonalcoholic steatohepatitis and compensated cirrhosis. Clin Gastroenterol Hepatol. 2021;19(6):1292–1293.e3. doi:10.1016/j.cgh.2020.06.070 Mikolasevic I, Orlic L, Zaputovic L, et al. Usefulness of liver test and controlled attenuation parameter in detection of nonalcoholic fatty liver disease in patients with chronic renal failure and coronary heart disease. Wien Klin Wochenschr. 2015;127(11–12):451–458. doi:10.1007/ s00508-015-0757-z Foucher J, Chanteloup E, Vergniol J, et al. Diagnosis of cirrhosis by transient elastography (FibroScan): a prospective study. Gut. 2006;55(3):403–408. doi:10.1136/ gut.2005.069153 Wong VW, Vergniol J, Wong GL, et al. Diagnosis of fbrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease. Hepatology. 2010;51(2):454–462. doi:10.1002/hep.23312 Eddowes PJ, Sasso M, Allison M, et al. Accuracy of FibroScan controlled attenuation parameter and liver stiffness measurement in assessing steatosis and fbrosis in patients with nonalcoholic fatty liver disease. Gastroenterology. 2019;156(6):1717–1730. doi:10.1053/j. gastro.2019.01.042 Takemoto R, Nakamuta M, Aoyagi Y, et al. Validity of FibroScan values for predicting hepatic fbrosis stage in patients with chronic HCV infection. J Dig Dis. 2009;10(2):145–148. doi:10.1111/j.1751-2980.2009.00377.x Loomba R, Wolfson T, Ang B, et al. Magnetic resonance elastography predicts advanced fbrosis in patients with nonalcoholic fatty liver disease: a prospective study. Hepatology. 2014;60(6):1920–1928. doi:10.1002/ hep.27362 Xiao H, Shi M, Xie Y, Chi X. Comparison of diagnostic accuracy of magnetic resonance elastography and Fibroscan for detecting liver fbrosis in chronic hepatitis B patients: a systematic review and meta-analysis. PLoS ONE. 2017;12(11):e0186660. doi:10.1371/journal. pone.0186660 Wentworth BJ, Caldwell SH. Pearls and pitfalls in nonalcoholic fatty liver disease: tricky results are common. Metabolism and Target Organ Damage. 2021;1(1):2. Furlan A, Tublin ME, Yu L, Chopra KB, Lippello A, Behari J. Comparison of 2D shear wave elastography, transient elastography, and MR elastography for the 63

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66.

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74.

64

diagnosis of fbrosis in patients with nonalcoholic fatty liver disease. AJR Am J Roentgenol. 2020;214(1):W20–26. doi:10.2214/ajr.19.21267 Davyduke T, Tandon P, Al-Karaghouli M, Abraldes JG, Ma MM. Impact of Implementing a “FIB-4 frst” strategy on a pathway for patients with NAFLD referred from primary care. Hepatol Commun. 2019;3(10):1322–1333. doi:10.1002/hep4.1411 Srivastava A, Gailer R, Tanwar S, et al. Prospective evaluation of a primary care referral pathway for patients with non-alcoholic fatty liver disease. J Hepatol. 2019;71(2):371–378. doi:10.1016/j.jhep.2019.03.033 Tapper EB, Hunink MG, Afdhal NH, Lai M, Sengupta N. Cost-effectiveness analysis: risk stratifcation of nonalcoholic fatty liver disease (NAFLD) by the primary care physician using the NAFLD fbrosis score. PLoS ONE. 2016;11(2):e0147237. doi:10.1371/journal.pone.0147237 Vilar-Gomez E, Lou Z, Kong N, Vuppalanchi R, Imperiale TF, Chalasani N. Cost effectiveness of different strategies for detecting cirrhosis in patients with nonalcoholic fatty liver disease based on United States health care system. Clin Gastroenterol Hepatol. 2020;18(10):2305–2314.e12. doi:10.1016/j.cgh.2020.04.017 Congly SE, Shaheen AA, Swain MG. Modelling the cost effectiveness of non-alcoholic fatty liver disease risk stratifcation strategies in the community setting. PLoS ONE. 2021;16(5):e0251741. doi:10.1371/journal. pone.0251741 Asphaug L, Thiele M, Krag A, Melberg HO. Costeffectiveness of noninvasive screening for alcoholrelated liver fbrosis. Hepatology. 2020;71(6):2093–2104. doi:10.1002/hep.30979 EASL-EASD-EASO clinical practice guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016;64(6):1388–1402. doi:10.1016/j.jhep.2015.11.004 Pandyarajan V, Gish RG, Alkhouri N, Noureddin M. Screening for nonalcoholic fatty liver disease in the primary care clinic. Gastroenterol Hepatol (NY). 2019;15(7):357–365. Newsome PN, Cramb R, Davison SM, et al. Guidelines on the management of abnormal liver blood tests. Gut. 2018;67(1):6–19. doi:10.1136/gutjnl-2017-314924

75. Dokmak A, Lizaola-Mayo B, Trivedi HD. The impact of nonalcoholic fatty liver disease in primary care: a population health perspective. Am J Med. 2021;134(1):23–29. doi:10.1016/j.amjmed.2020.08.010 76. Ando Y, Jou JH. Nonalcoholic fatty liver disease and recent guideline updates. Clin Liver Dis (Hoboken). 2021;17(1):23–28. doi:10.1002/cld.1045 77. Siddiqui MS, Yamada G, Vuppalanchi R, et al. Diagnostic accuracy of noninvasive fbrosis models to detect change in fbrosis stage. Clin Gastroenterol Hepatol. 2019;17(9):1877–1885.e5. doi:10.1016/j. cgh.2018.12.031 78. McPherson S, Stewart SF, Henderson E, Burt AD, Day CP. Simple non-invasive fbrosis scoring systems can reliably exclude advanced fbrosis in patients with nonalcoholic fatty liver disease. Gut. 2010;59(9):1265–1269. doi:10.1136/gut.2010.216077 79. Sun W, Cui H, Li N, et al. Comparison of FIB-4 index, NAFLD fbrosis score and BARD score for prediction of advanced fbrosis in adult patients with non-alcoholic fatty liver disease: a meta-analysis study. Hepatol Res. 2016;46(9):862–870. doi:10.1111/hepr.12647 80. Younossi ZM, Felix S, Jeffers T, et al. Performance of the enhanced liver fbrosis test to estimate advanced fbrosis among patients with nonalcoholic fatty liver disease. JAMA Netw Open. 2021;4(9):e2123923. doi:10.1001/ jamanetworkopen.2021.23923 81. Mózes FE, Lee JA, Selvaraj EA, et al. Diagnostic accuracy of non-invasive tests for advanced fbrosis in patients with NAFLD: an individual patient data meta-analysis. Gut. 2021. doi:10.1136/ gutjnl-2021-324243 82. Papatheodoridi M, Hiriart JB, Lupsor-Platon M, et al. Refning the Baveno VI elastography criteria for the defnition of compensated advanced chronic liver disease. J Hepatol. 2021;74(5):1109–1116. doi:10.1016/j. jhep.2020.11.050 83. de Franchis R. Expanding consensus in portal hypertension: report of the Baveno VI consensus workshop: stratifying risk and individualizing care for portal hypertension. J Hepatol. 2015;63(3):743–752. doi:10.1016/j.jhep.2015.05.022

7 ULTRASOUND-BASED TECHNIQUES IN NAFLD

7 Ultrasound-Based Techniques in NAFLD Vikas Taneja, Nezam H. Afdhal and Michelle J. Lai CONTENTS 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 7.2 Description of Ultrasound-Based Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 7.2.1 Vibration-Controlled Transient Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 7.2.2 Shear Wave Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 7.2.3 Acoustic Radiation Force Imaging (ARFI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 7.3 Steatosis Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 7.3.1 Performance of Conventional Ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 7.3.2 Performance of VCTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 7.4 Differentiation of Simple Steatosis from NASH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 7.5 Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 7.5.1 Performance of VCTE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 7.5.2 Comparison among M and XL Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 7.5.3 Performance of SWE and ARFI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 7.5.4 Comparison among VCTE, SWE and ARFI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 7.5.5 Combination of VCTE with Serum Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 7.5.6 Comparisons with Other Modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 7.6 Limitations of Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 7.7 Portal Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Abbreviations ARFI: acoustic radiation force impulse CAP: controlled attenuation parameter CSPH: clinically signifcant portal hypertension HVPG: hepatic venous pressure gradient LSM: liver stiffness measurement NAFLD: nonalcoholic fatty liver disease NASH: nonalcoholic steatohepatitis VCTE: vibration-controlled transient elastography ROI: region of interest SWE: shear wave elastography 7.1 INTRODUCTION While liver biopsy has remained the gold standard for evaluation of NAFLD, its use is limited by invasive risk, sampling error, cost and low patient acceptance.1 Consequently, the use of noninvasive modalities, such as ultrasound- (US-) based techniques for diagnosis of NAFLD has evolved rapidly in the last few years to become the standard of care.2 Abdominal US, which is commonly utilized as a screening test for elevated liver enzymes was one of the earliest diagnostic modality to be used for noninvasive evaluation of NAFLD. It is widely available, well-tolerated and relatively inexpensive. However, US is not sensitive (cannot detect mild steatosis), can be confounded by other factors that increase hepatic echogenicity and does not offer quantitative assessment or assessment of infammation or fbrosis. These limitations have resulted in the development of ultrasound-based elastographic techniques to better quantify hepatic fbrosis and fat and more recently to try to differentiate simple steatosis from nonalcoholic steatohepatitis (NASH). Initially the use of elastography was developed for the diagnosis of fbrosis in viral hepatitis C but now has emerged as one of the most accurate noninvasive methods of assessment of both fbrosis and steatosis in patients with NAFLD.3 Subsequently, current guidelines recommend utilizing transient elastography for noninvasive evaluation of fbrosis in patients with NAFLD.2 DOI: 10.1201/9781003386698-9

In this chapter, we will examine the current use of elastographic techniques for diagnosing liver steatosis and fbrosis in NAFLD and differentiating simple steatosis from NASH. 7.2 DESCRIPTION OF ULTRASOUNDBASED TECHNOLOGIES In addition to standard 3D ultrasound, currently several newer elastographic technologies are available for the evaluation of fat and fbrosis in NAFLD, including vibrationcontrolled transient elastography (VCTE), shear wave elastography (SWE) and acoustic radiation force impulse (ARFI) elastography. All three techniques share the same principle of estimating liver stiffness through measurement of elastic modulus; i.e., the stiffer the tissue is, the faster the shear wave velocity will be. While VCTE has been extensively studied in the setting of well-designed studies with paired histology leading to established cutoffs and quality criteria, the choice of technique is also infuenced by local availability and expertise (Table 7.1). 7.2.1 Vibration-Controlled Transient Elastography Ultrasound propagation through liver parenchyma is attenuated to varying degrees in the presence of steatosis. Controlled attenuation parameter (CAP) is a measure that estimates hepatic steatosis by capturing the extent of such attenuation. Vibration-controlled transient elastography (VCTE) (FibroScan®, Echosens, Paris, France) was approved by the United States Food and Drug Administration (FDA) in 2013. The device can measure CAP and liver stiffness (a surrogate for fbrosis, measured in kPa) simultaneously. The machine was initially introduced with a standardsized probe (M) (3.5 MHz, 2 mm vibration amplitude). The performance of the M probe was limited by high rates of failure in obese patients of about 20%,4 but subsequently, with the introduction of an XL probe (2.5 MHz, 3 mm vibration amplitude) and introduction of a software to automatically determine the choice of probe, the failure rates have improved to less than 5%.5 65

SECTION II: DIAGNOSTIC TESTS

Table 7.1: Characteristics of Elastography Techniques for Evaluation of Fibrosis in NAFLD Technique

Strength of Evidence

Cost

VCTE SWE

+++ ++

++ ++

ARFI

++

++

Performance

Quality Criteria

Limitations

+++ +++ (F4) ++ (F1, F2 and F3) +++

Standardized Individual operator dependent

Requires a dedicated device. Elimination of artifacts during ROI placement requires attention/optimal technique. Not enough evidence to establish intraobserver agreement.1

Individual operator dependent

+: Low; ++: Moderate; +++: High 1

Ferraioli G, Filice C, Castera L, et al. WFUMB Guidelines and Recommendations for Clinical Use of Ultrasound Elastography: Part 3: Liver. Ultrasound in Medicine & Biology. 2015;41(5):1161–1179. doi:10.1016/j.ultrasmedbio.2015.03.007

VCTE utilizes an automated movement of the ultrasound transducer to generate a brief push (“thump”), which propagates a shear wave. The probe is placed within the 9th–11th intercostal space, and the shear wave is evaluated by the receiver at a fxed distance.6 CAP values are expressed as dB/m and range from 100 dB/m to 400 dB/m. As compared to a liver biopsy, CAP is much more convenient as it provides immediate result and is less prone to sampling error.7 Additionally, CAP has good interobserver agreement, which makes it a valuable tool for longitudinal follow-up of patients.8 TE values in a healthy population range from 4.4 kPa to 5.5 kPa,9,10 with higher values in males as compared to females.11 Values are not affected by age.12 The following criteria are utilized to establish the validity of a result:13 (1) at least 10 valid measurements; (2) a success rate (the ratio of valid measurements to the total number of measurements) above 60%; and (3) an interquartile range (IQR) less than 30% of the median LS measurements.14 The mechanical impulse is aborted if the probe fails to detect liver parenchyma (e.g., if the probe lies over the rib). 7.2.2 Shear Wave Elastography SWE targets a region of interest (ROI) in the liver using acoustic impulses, and the shear wave speed is measured to generate liver stiffness measurement (LSM) in kPa. The operator defnes a vessel-free region using conventional B mode ultrasound, and a series of push pulses are utilized to create a plane of shear waves. After generating the shear wave, the device switches to radio frequency imaging mode to capture the shear wave velocity.15 Subsequently, tissue stiffness is calculated by the formula E = pc2, where E is the tissue elasticity (in kPa), p is the tissue density and c is the shear wave speed.16 The technique has an advantage of being an existing feature on some of the US machines. 7.2.3 Acoustic Radiation Force Imaging (ARFI) ARFI is a technique that utilizes focused acoustic energy to provide mechanical excitation directly to the tissue of interest and subsequently generating shear waves in vivo. The tissue in the region of interest is excited mechanically using short duration acoustic pulses with a frequency of 2.67 MHz to generate tissue displacement, which results in shear wave propagation away from the area of excitation. The speed of the shear wave away from the region of excitation (ROE) is used to estimate shear moduli (measured in kPa).17 This unique mechanism circumvents the challenge associated with propagation of external mechanical excitation into the liver tissue. Notably, quality criteria for 2D SWE and ARFI remain to be established in large-scale 66

studies, and most studies on their use have utilized quality criteria similar to the criteria for VCTE.18–21 7.3 STEATOSIS MEASUREMENT 7.3.1 Performance of Conventional Ultrasound Steatosis leads to increased echogenicity of the hepatic parenchyma due to closely spaced, fne echoes. The echogenicity of normal hepatic parenchyma is equal to or slightly greater than that of the renal cortex.22 In the presence of enough fat (>30%), the echogenicity of the liver may exceed that of the kidney and spleen, a feature called “bright liver,” which is a commonly used diagnostic feature. Additionally, the presence of liver fat reduces the ability of the US beam to penetrate the hepatic parenchyma.22 This leads to loss of defnition of diaphragm and posterior darkness, described as “posterior beam attenuation.”23 Focal fat deposition or focal fat sparing may be challenging to identify with US; however, it can be identifed by the localized presence of the preceding imaging characteristics. Features such as poorly delineated margins and absence of mass effect are helpful in distinguishing focal fat deposition or sparing from mass lesions, but frequently an MRI is necessary.22 The extent of steatosis is commonly reported as mild, moderate or severe; some studies have utilized scoring systems to mirror the histological classifcation.24 The sensitivity and specifcity of US for detection of steatosis are dependent on the severity of steatosis, with higher accuracy for moderate (25–50%) to severe (>50%) steatosis. A meta-analysis based on 19 studies noted a sensitivity of 73.3% (95% CI 62.2–82.1%) and specifcity of 84.4 (76.2–90.1) for any steatosis (>0% steatosis on biopsy).26 However, the performance of US was improved in patients with higher grades of steatosis. The sensitivity and specifcity in patients with moderate steatosis (25–50% steatosis) were 85.7% (95% CI 78.4–90.8%) and 85.2% (95% CI 76.9–90.9%), and in those with severe steatosis (>50% steatosis), they were 91.1% (95% CI 63.0–98.4%) and 91.9% (95% CI 74.3–97.8%), respectively. The overall sensitivity and specifcity for detecting moderate-severe steatosis on US was reported to be 84.8% (95% CI 79.5–88.9%) and 93.6% (95% CI 87.2–97.0%), respectively, in a meta-analysis that included 49 studies (4,720 participants).25 Positive likelihood ratio and negative likelihood ratio were 13.3 (6.4–27.6), and 0.16 (0.12–0.22), respectively. Area under the receiver operating characteristic curve (AUROC) was 0.931 (95% CI: 0.91–0.95%). For detection of milder steatosis (≥10% steatosis), the specifcity was reduced to 88% (95% CI 63–97%); however, sensitivity was maintained at 93% (88–97%). (Additionally, this study compared sensitivity

7 ULTRASOUND-BASED TECHNIQUES IN NAFLD

and specifcity of various parameters such as liver to kidney contrast, vessel wall brightness, and deep beam attenuation for detection of steatosis. However, it included NAFLD and non-NAFLD patients.) A few studies have evaluated the use of US for quantitative estimation of steatosis, such as hepatorenal index27 and far-feld slope (FFS) algorithm,28 which estimates the extent of deep beam attenuation to quantify steatosis. However, such methods have not yet been validated in larger cohorts. Limitations of US include operator dependency, inability to detect S0, >S1 and >S2, respectively.31 However, this study included a high proportion of patients (80.4%) with other liver diseases such as chronic viral hepatitis, who may have undergone liver biopsy for indications other than NAFLD. Additionally, the study was conducted prior to the introduction of the XL probe and consequently only included

data from the M probe (BMI ≥35 was an exclusion criterion). The AUCROC for the presence of hepatic steatosis as compared to liver biopsy as a reference was 0.82. A more recent meta-analysis by Petroff et al.32 (930 patients: XL probe, 1,274 patients: M probe) reported the overall cutoffs of 294 dB/m (95% CI 286–313 dB/m), 310 dB/m (95% CI 305–321 dB/m) and 331 dB/m (95% CI 319–340 dB/m) for diagnosing steatosis grades >S0, >S1 and >S2, respectively. The corresponding cutoffs for the XL probe were 297 dB/m (95% CI 287–323 dB/m), 317 dB/m (95% CI 306–334 dB/m) and 333 dB/m (95% CI 320–340 dB/m), respectively. Notably, in order to optimize the sensitivity and specifcity of the estimates, the authors used the Youden approach to determine the cutoffs. If instead of the Youden approach, the sensitivity was set at 90%, the overall cutoffs (M and XL probes) were 263 dB/m (95% CI 256–270 dB/m), 286 dB/m (95% CI 282–292 dB/m) and 297 dB/m (95% CI 286–307 dB/m) for steatosis grades >S0, >S1 and >S2, respectively. The AUC for diagnosing steatosis grades >S0, >S1 and >S2 were 0.82, 0.75 and 0.71, respectively (Table 7.2). In our clinical practice, we use a cutoff of 285 dB/m for steatosis and 330 dB/m for severe steatosis. 7.4 DIFFERENTIATION OF SIMPLE STEATOSIS FROM NASH The biopsy characteristics of NASH include infammation, apoptosis (balloon degeneration) and fbrosis, and a key component of NAFLD management lies in identifcation of the subgroup of patients who develop NASH and fbrosis. Few studies have reported the diagnostic utility of TE in detecting NASH. The largest such study included 183 patients undergoing liver biopsy and concomitant TE for suspected NAFLD. A scoring system that incorporated

Table 7.2: Performance of Controlled Attenuation Parameter in Studies among Patients with NAFLD Using Histology as Reference Stage of Steatosis

Cutoff (dB/m)

Sensitivity (%)

≥S1

236 261 270 285 270 305 310 301 306 311 312

82 72 84 80 78 63 79 76 80 87 64

≥S2

S3

1

2

3

4

5

6

Specifcity (%)

Positive Predictive Value (%)

Negative Predictive Value (%)

91 86 82 77 81 69 71 68 40 47 70

99 98 NR 99 73 56 86 NR 32 43 26

67 23 NR 16 76 75 59 NR 85 88 92

References Imajo1 Park2 Enooku3 Siddiqui4 Imajo1 Park2 De Ledinghen5 Friedrich-Rust6 Siddiqui4 De Ledinghen5 Park2

Imajo K, Kessoku T, Honda Y, et al. Magnetic resonance imaging more accurately classifes steatosis and fbrosis in patients with nonalcoholic fatty liver disease than transient elastography. Gastroenterology. 2016;150(3):626–637.e7. doi:10.1053/j.gastro.2015.11.048 Park CC, Nguyen P, Hernandez C, et al. Magnetic resonance elastography vs transient elastography in detection of fbrosis and noninvasive measurement of steatosis in patients with biopsy-proven nonalcoholic fatty liver disease. Gastroenterology. 2017;152(3):598–607.e2. doi:10.1053/j.gastro.2016.10.026 Enooku K, Tateishi R, Fujiwara N, et al. 1330 Non-invasive measurement of liver steatosis by controlled attenuation parameter (CAP) using FibroScan® in patients with nonalcoholic fatty liver disease (NAFLD). Journal of Hepatology. 2013; Supplement 1(58):S536–S537. doi:10.1016/S0168–8278(13)61330–6 Siddiqui MS, Vuppalanchi R, Van Natta ML, et al. Vibration-controlled transient elastography to assess fbrosis and steatosis in patients with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 2019;17(1):156–163.e2. doi:10.1016/j.cgh.2018.04.043 de Lédinghen V, Wong GLH, Vergniol J, et al. Controlled attenuation parameter for the diagnosis of steatosis in non-alcoholic fatty liver disease. J Gastroenterol Hepatol. 2016;31(4):848–855. doi:10.1111/jgh.13219 Friedrich-Rust M, Romen D, Vermehren J, et al. Acoustic radiation force impulse-imaging and transient elastography for non-invasive assessment of liver fbrosis and steatosis in NAFLD. Eur J Radiol. 2012;81(3):e325–331. doi:10.1016/j.ejrad.2011.10.029

67

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CAP and LSM in addition to ALT values was able to identify patients with NASH with AUROC of 0.812 (95% CI 0.724–0.880%).33 The prevalence of NASH in this study was 51.6%. In another study of 47 patients that underwent transient elastography withing 2 weeks of liver biopsy, TE was noted to have an AUC of 0.82 (0.70–0.94) for diagnosis of NASH vs. simple steatosis.34 The role of SWE and ARFI in diagnosing NASH also remains unclear owing to a paucity of large-scale studies. In a single-center study of prospectively enrolled 102 patients with biopsy-proven NAFLD, AUC for diagnosis of lobular infammatory activity, as assessed by a shear wave dispersion slope, for grades >I0, >I1, >I2, were 0.89, 0.85 and 0.78, respectively.35 However, only about 8% of the patients had advanced fbrosis or cirrhosis. Similarly, only one study has evaluated the use of ARFI in NASH.36 Among 64 biopsy-proven NAFLD, ARFI had AUC of 0.86 in discriminating NASH from simple steatosis. At a cutoff of ARFI velocity >1.1 m/s, the sensitivity and specifcity of identifying NASH were 77 and 72%, respectively (positive predictive value 85%, negative predictive value 60%). The study was limited by a small sample size, and some of the patients in the study underwent liver biopsy as long as 6 months before ARFI elastography, thus limiting the validity of the fndings. 7.5 FIBROSIS Fibrosis in the liver alters liver stiffness, which can be measured by elastography. Elastography utilizes US to measure tissue shear deformations resulting from an externally applied force, such as acoustic vibration or probe palpation.40 Three techniques as just described are available: TE, SWE and ARFI. The importance of staging fbrosis in NAFLD cannot be underestimated since fbrosis has the strongest correlation with clinical liver outcomes including morbidity and mortality.41– 43 Fibrosis is also one of the hallmarks of progressive NASH, and therefore diagnosing fbrosis greater than F2 is diagnostic of probable NASH when combined with clinical risk factors such as obesity and diabetes and elevated ALT. 7.5.1 Performance of VCTE In a systematic review involving 1,047 patients across 9 studies, the diagnostic accuracy of TE was satisfactory for F3 (85% sensitivity, 82% specifcity) and cirrhosis (92% sensitivity, 92% specifcity), respectively. However, the accuracy dropped to 79% sensitivity, 75% specifcity for F2 fbrosis.44 In a recent meta-analysis that included up to 4,219 patients who underwent VCTE (both M and XL probes) and liver biopsy, the AUC, sensitivity and specifcity of VCTE in diagnosing any fbrosis (≥F1) was 0.82 (95% CI 0.78–0.85%), 78% (95% CI 73–82%), 72% (95% CI 65–79%). The performance of VCTE improved for the diagnosis of advanced fbrosis (≥F3) and cirrhosis (F = 4), with AUC, sensitivity and specifcity of 0.85 (95% CI 0.83–0.87%), 80% (95% CI 77–83%), 77% (95% CI 74–80%) for advanced fbrosis and 0.89 (95% CI 0.84–0.93%), 76% (95% CI 70–82%), 88% (95% CI 85–91%) for cirrhosis, respectively.45 Estimates of optimal cutoffs for LSM by VCTE for the diagnosis of various stages of fbrosis for maximal sensitivity and specifcity vary from 7.2 to 11.4 kPa (Table 7.3). In a study involving 246 patients who underwent liver stiffness measurement and liver biopsy, at a cutoff value of

68

7.9 kPa, the sensitivity, specifcity, and positive and negative predictive values for F3 or greater disease were 91, 75, 52, and 97, respectively.46 The negative predictive value of LSM F1 fbrosis were 0.86 (0.78–0.90), 69% (59–77%), 85% (80–88%); for diagnosing >F2 fbrosis were 0.89 (0.83–0.95), 80% (70–88%), 86% (82–92%); and for diagnosing >F3 fbrosis were 0.90 (0.82–0.95), 76% (59–87%), 88% (82–92%), respectively (Tables 7.4 and 7.5). 7.5.4 Comparison among VCTE, SWE and ARFI Few studies have compared TE, SWE and ARFI in the same cohort of patients. In a French cohort of prospectively enrolled 291 patients with biopsy proven NAFLD, Cassinotto et al. noted comparable performance among TE, ARFI and 2D SWE.19 The AUC for SWE, FibroScan®, and ARFI were 0.86, 0.82, and 0.77 for diagnoses of F2; 0.89,

0.86, and 0.84 for F3; and 0.88, 0.87, and 0.84 for F4, respectively. Obesity (BMI ≥ 30 kg/m2, waist circumference≥ 102 cm or increased parietal wall thickness) was associated with unreliable results in ARFI and with LSM failures in TE and SWE. However, overall, there were no differences in the reliability of results, with 79.7% reliable results produced by SWE, 76.6% by FibroScan®, and 81% by ARFI (p values for all comparisons were nonsignifcant). 7.5.5 Combination of VCTE with Serum Markers In a study of 139 biopsy-proven NAFLD patients, a combination of LSM with Fib-4 or NFS increased the accuracy of fbrosis detection in NAFLD, yielding a positive predictive value of 0.735 at a sensitivity of 89% and a negative predictive value of 0.932 at a specifcity of 82%.50 A combination of LSM by VCTE, platelet count, diabetes, ALT-toAST ratio, gender and age (AGILE 3+) has demonstrated a more optimal positive predictive value compared to

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Table 7.4: Performance of SWE in Studies among Patients with NAFLD Using Histology as Reference Stage of Fibrosis

Cutoff (kPa)

Sensitivity (%)

Specifcity (%)

≥F2

8.3 8.4 8.7 9.3 10.7 14.4 15.1 15.7

87 77 71 84 90 59 90 100

55 66 90 70 61 90 78 82

≥F3 F4

1

2

3

4

References Lee1 Ozturk3 Cassinotto 20162 Ozturk3 Lee1 Cassinotto 20162 Lee1 Takeuchi4

Lee MS, Bae JM, Joo SK, et al. Prospective comparison among transient elastography, supersonic shear imaging, and ARFI imaging for predicting fbrosis in nonalcoholic fatty liver disease. PLOS ONE. 2017;12(11):e0188321. doi:10.1371/journal.pone.0188321 Cassinotto C, Boursier J, de Lédinghen V, et al. Liver stiffness in nonalcoholic fatty liver disease: A comparison of supersonic shear imaging, FibroScan, and ARFI with liver biopsy. Hepatology. 2016;63(6):1817–1827. doi:10.1002/hep.28394 Ozturk A, Mohammadi R, Pierce TT, et al. diagnostic accuracy of shear wave elastography as a non-invasive biomarker of high-risk nonalcoholic steatohepatitis in patients with non-alcoholic fatty liver disease. Ultrasound in Medicine & Biology. 2020;46(4):972–980. doi:10.1016/j.ultrasmedbio.2019.12.020 Takeuchi H, Sugimoto K, Oshiro H, et al. Liver fbrosis: noninvasive assessment using supersonic shear imaging and FIB4 index in patients with non-alcoholic fatty liver disease. J Med Ultrason (2001). 2018;45(2):243–249. doi:10.1007/s10396-017-0840-3

Table 7.5: Performance of ARFI in Studies among Patients with NAFLD Using Histology as Reference Stage of Fibrosis

Cutoff (m/s)

Sensitivity (%)

Specifcity (%)

≥F1

1.105 1.29 1.165 1.17 1.32 1.42 1.45 1.48 1.635 1.75 1.89

77 54 85 86 56 97 76 86 92 74 90

71 77 90 87 91 97 68 95 92 67 95

≥F2

≥F3

F4

1

2

3

4

5

AUC NR 0.66 0.94 0.90 0.77 0.99 91 0.98 0.98 0.91 0.98

References Fierbinteau2 Cui4 Fierbinteau3 Attia5 Cassinotto 20162 Attia5 Friedrich-Rust1 Fierbinteau3 Fierbinteau3 Friedrich-Rust1 Attia5

Friedrich-Rust M, Romen D, Vermehren J, et al. Acoustic radiation force impulse imaging and transient elastography for non-invasive assessment of liver fbrosis and steatosis in NAFLD. Eur J Radiol. 2012;81(3):e325–331. doi:10.1016/j.ejrad.2011.10.029 Cassinotto C, Boursier J, de Lédinghen V, et al. Liver stiffness in nonalcoholic fatty liver disease: a comparison of supersonic shear imaging, FibroScan, and ARFI with liver biopsy. Hepatology. 2016;63(6):1817–1827. doi:10.1002/hep.28394 Fierbinteanu Braticevici C, Sporea I, Panaitescu E, Tribus L. Value of acoustic radiation force impulse imaging elastography for non-invasive evaluation of patients with nonalcoholic fatty liver disease. Ultrasound in Medicine & Biology. 2013;39(11):1942–1950. doi:10.1016/j. ultrasmedbio.2013.04.019 Cui J, Heba E, Hernandez C, et al. Magnetic resonance elastography is superior to acoustic radiation force impulse for the diagnosis of fbrosis in patients with biopsy-proven nonalcoholic fatty liver disease: a prospective study. Hepatology. 2016;63(2):453–461. doi:10.1002/hep.28337 Attia D, Bantel H, Lenzen H, Manns MP, Gebel MJ, Potthoff A. Liver stiffness measurement using acoustic radiation force impulse elastography in overweight and obese patients. Alimentary Pharmacology & Therapeutics. 2016;44(4):366–379. doi:10.1111/apt.13710

LSM alone. More recently, an improved version of this combination (AGILE 4)51 was shown to reduce the percentage of patients with indeterminate results to 13%, as compared to 21% for VCTE. The AGILE4 score achieved an AUC of 0.93 (95% CI 0.91–0.96%) and outperformed LSM alone (AUC 0.89, 95% CI 0.86–0.93%) and FIB4 score (AUC 0.83, 95% CI 0.79–0.88%). In order to address the challenge of identifying patients that are at the greatest risk of NAFLD progression, a combination of LSM by VCTE, CAP and AST, called the FibroScan® aspartate aminotransferase (FAST) score, has been developed.52 The score has demonstrated satisfactory performance for diagnosis of NASH (using the FLIP defnition) with NAS 4 or higher and fbrosis stage 2 or higher with a PPV of 0·83 NPV of 0·85. The score was initially derived from a cohort of 70

350 prospectively enrolled patients and subsequently validated in seven cohorts of a total of 1,026 patients. 7.5.6 Comparisons with Other Modalities In a meta-analysis of 13,294 patients, CAP-TE, SWE and MRE were superior than lab tests (NFS, BARD, FIB-4, APRI) in diagnosing fbrosis.53 SWE was similar in accuracy to MRE for detection of the following stages of fbrosis– signifcant fbrosis (F2–F4), advanced fbrosis (F3–F4) and cirrhosis (F4). The sensitivities and specifcities of the FibroScan® M (threshold of 8.7–9), SWE and MRE for detecting advanced fbrosis were 0.87 and 0.79, 0.90 and 0.93, and 0.84 and 0.90, respectively. The summary AUROC values using the FibroScan® M probe, XL probe, SWE, and MRE for diagnosing advanced fbrosis were 0.88, 0.85, 0.95, and

7 ULTRASOUND-BASED TECHNIQUES IN NAFLD

0.96, respectively. The authors noted that SWE and MRE were statistically better than VCTE in identifying advanced fbrosis. However, in the meta-analysis by Selvaraj et al., which included studies on ARFI in addition, the authors concluded that in the setting of a reliable LSM, all three US-based modalities (VCTE, SWE and ARFI) had acceptable diagnostic accuracy for advanced fbrosis and cirrhosis. When minimum acceptable criteria were defned as greater than 80% for both sensitivity and specifcity, ARFI and MRE met these criteria for diagnosis of advanced fbrosis, and only MRE met these criteria for diagnosis of cirrhosis. 7.6 LIMITATIONS OF ELASTOGRAPHY Since all three techniques are based on the principle of measuring tissue elasticity through shear wave propagation, they share a majority of limitations that have been most extensively studied in VCTE. The propagation of shear wave across fuid is inconsistent, and subsequently LSM values in patients with ascites are unreliable. Although the failure rates in patients with obesity have been largely mitigated by the introduction of the XL probe, extreme obesity with skin-to-liver capsule distance > 3.4 cm (BMI > 40 kg/m2) may still be associated with unreliable estimates up to 15%.54 Liver stiffness is also increased with hepatic infammation (ALT/AST >5 ULN), obstructive cholestasis, alcoholic hepatitis, amyloidosis, lymphomas and extramedullary hematopoiesis. Furthermore, enlarged veins damp shear stress, thus affecting the accuracy of LSM measurement in hepatic congestion (CHF). 7.7 PORTAL HYPERTENSION LSM as measured by VCTE has been shown to corelate with hepatic venous pressure gradient (HVPG). In a study of 150 patients who underwent liver biopsy with hemodynamic measurements, LSM had AUC of 0.94 (95% CI 0.90–0.98%) for diagnosing clinically signifcant portal hypertension (CSPH), defned as HVPG ≥10 mmHg.55 At a cutoff value of >21 kPa, the sensitivity and specifcity for identifying CSPH were 89.9 and 93.2%, respectively. The role of spleen stiffness has also been evaluated in identifying patients with PHT. The increased spleen stiffness in advanced chronic liver disease is hypothesized to be due to increased splenic congestion and, to some extent, splenic fbrosis.56 Subsequently, while LSM identifes liver fbrosis burden, SS may be more refective of downstream hemodynamic consequences.57 In a study of 113 patients with cirrhosis due to hepatitis C, Colecchia et al.58 noted a good correlation between SS and HVPG. Patients with PHT had higher SS than patients without PHT (59kPa vs. 39 kPa) when CSPH was defned as HVPG> 10 mmHg, and a model that incorporated LSM and SS achieved a robust R2 of 0.82 for prediction of HVPG. While CSPH is a hemodynamic measure related to HVPG, due to a suffciently large body of evidence demonstrating the accuracy of LSM in the assessment of hepatic fbrosis which is the primary driver of HVPG, recent guidelines recommend utilizing LSM to identify patients that are at high or low risk of CSPH as outlined in the Baveno VII criteria.59 Whereas LSM by TE ≤15 kPa plus platelet count ≥150×109/L rules out CSPH (sensitivity and negative predictive value >90%) (LOE B, weak recommendation), identifcation of patients at high risk of CSPH may be accomplished by utilizing the ANTICIPATE model for nonobese NASHrelated cACLD60 (LOE B, weak recommendation) or the ANTICIPATE-NASH model for NASH-related cACLD61 (LOE C, weak recommendation).

In conclusion, ultrasound-based technologies that utilize elastography has become the standard of care for noninvasive diagnosis and staging of liver fbrosis in NAFLD. While VCTE is the best characterized modality, there is growing literature to support the use of SWE and ARFI, which have demonstrated comparable diagnostic accuracy and reliability. However, quality criteria and prespecifed thresholds remain to be established for SWE and ARFI. Additionally, the role of elastography in the evaluation of NASH remains an area of active research to identify patients for enrollment in clinical trials for treatment of NASH without needing a liver biopsy. Future studies to evaluate the longitudinal association of change in fbrosis severity on elastography and risk of development of hepatic decompensation (increase in LSM) or improvement in fbrosis (reduction in LSM) would be crucial to assess the treatment response when pharmacological therapy becomes available. REFERENCES 1. Ratziu V, Charlotte F, Heurtier A, et al. Sampling variability of liver biopsy in nonalcoholic fatty liver disease. Gastroenterology. 2005;128(7):1898–1906. 2. European Association for the Study of the Liver. Electronic address: [email protected], Clinical Practice Guideline Panel, Chair: EASL Governing Board representative: Panel members: EASL Clinical Practice Guidelines on non-invasive tests for evaluation of liver disease severity and prognosis—2021 update. J Hepatol. 2021;75(3):659–689. doi:10.1016/j.jhep.2021.05.025 3. Tapper EB, Loomba R. Noninvasive imaging biomarker assessment of liver fbrosis by elastography in NAFLD. Nat Rev Gastroenterol Hepatol. 2018;15(5):274–282. doi:10.1038/nrgastro.2018.10 4. Castéra L, Foucher J, Bernard PH, et al. Pitfalls of liver stiffness measurement: a 5-year prospective study of 13,369 examinations. Hepatology. 2010;51(3):828–835. 5. Vuppalanchi R, Siddiqui MS, Van Natta ML, et al. Performance characteristics of vibration-controlled transient elastography for evaluation of nonalcoholic fatty liver disease. Hepatology. 2018;67(1):134–144. 6. Sandrin L, Fourquet B, Hasquenoph JM, et al. Transient elastography: a new noninvasive method for assessment of hepatic fbrosis. Ultrasound Med Biol. 2003;29(12):1705–1713. doi:10.1016/j. ultrasmedbio.2003.07.001 7. Lédinghen V de, Wong GLH, Vergniol J, et al. Controlled attenuation parameter for the diagnosis of steatosis in non-alcoholic fatty liver disease. J Gastroenterol Hepatol. 2016;31(4):848–855. 8. Ferraioli G, Tinelli C, Lissandrin R, et al. Interobserver reproducibility of the controlled attenuation parameter (CAP) for quantifying liver steatosis. Hepatol Int. 2014;8(4):576–581. 9. Colombo S, Belloli L, Zaccanelli M, et al. Normal liver stiffness and its determinants in healthy blood donors. Dig Liver Dis. 2011;43(3):231–236. doi:10.1016/j. dld.2010.07.008 10. Roulot D, Costes JL, Buyck JF, et al. Transient elastography as a screening tool for liver fbrosis and cirrhosis in a community-based population aged over 45 years. Gut. 2011;60(7):977–984. doi:10.1136/ gut.2010.221382 11. Corpechot C, El Naggar A, Poupon R. Gender and liver: is the liver stiffness weaker in weaker sex? Hepatology. 2006;44(2):513–514. doi:10.1002/hep.21306 71

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50.

and technology. Ultraschall Med. 2013;34(2):169–184. doi:10.1055/s-0033-1335205 Tada T, Kumada T, Toyoda H, et al. Progression of liver fbrosis is associated with non-liver-related mortality in patients with nonalcoholic fatty liver disease. Hepatol Commun. 2017;1(9):899–910. doi:10.1002/hep4.1105 Unalp-Arida A, Ruhl CE. Liver fbrosis scores predict liver disease mortality in the United States population. Hepatology. 2017;66(1):84–95. doi:10.1002/ hep.29113 Schonmann Y, Yeshua H, Bentov I, Zelber-Sagi S. Liver fbrosis marker is an independent predictor of cardiovascular morbidity and mortality in the general population. Dig Liver Dis. 2021;53(1):79–85. doi:10.1016/j.dld.2020.10.014 Kwok R, Tse YK, Wong GLH, et al. Systematic review with meta-analysis: non-invasive assessment of non-alcoholic fatty liver disease-the role of transient elastography and plasma cytokeratin-18 fragments. Aliment Pharmacol Ther. 2014;39(3):254–269. doi:10.1111/apt.12569 Selvaraj EA, Mózes FE, Jayaswal ANA, et al. Diagnostic accuracy of elastography and magnetic resonance imaging in patients with NAFLD: a systematic review and meta-analysis. J Hepatol. 2021;75(4):770–785. doi:10.1016/j.jhep.2021.04.044 Wong VWS, Vergniol J, Wong GLH, et al. Diagnosis of fbrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease. Hepatology. 2010;51(2):454–462. doi:10.1002/hep.23312 Wong VWS, Irles M, Wong GLH, et al. Unifed interpretation of liver stiffness measurement by M and XL probes in non-alcoholic fatty liver disease. Gut. 2019;68(11):2057–2064. Oeda S, Takahashi H, Imajo K, et al. Accuracy of liver stiffness measurement and controlled attenuation parameter using FibroScan® M/XL probes to diagnose liver fbrosis and steatosis in patients with nonalcoholic fatty liver disease: a multicenter prospective study. J Gastroenterol. 2020;55(4):428–440. Cardoso AC, Cravo C, Calçado FL, et al. The performance of M and XL probes of FibroScan for the diagnosis of steatosis and fbrosis on a Brazilian nonalcoholic fatty liver disease cohort. Eur J Gastroenterol Hepatol. 2020;32(2):231–238. Jafarov F, Kaya E, Bakir A, Eren F, Yilmaz Y. The diagnostic utility of fbrosis-4 or nonalcoholic fatty liver disease fbrosis score combined with liver stiffness measurement by fbroscan in assessment of advanced liver fbrosis: a biopsy-proven nonalcoholic fatty liver disease study. Eur J Gastroenterol Hepatol. 2020;32(5):642–649. doi:10.1097/ MEG.0000000000001573

51. Younossi ZM, Harrison SA, Newsome PN, et al. Improving diagnosis of cirrhosis in patients with NAFLD by combining liver stiffness measurement by vibration-controlled transient elastography and routine biomarkers: a global derivation and validation study. In: The Liver Meeting Digital ExperienceTM. AASLD. 52. Newsome PN, Sasso M, Deeks JJ, et al. FibroScan-AST (FAST) score for the non-invasive identifcation of patients with non-alcoholic steatohepatitis with signifcant activity and fbrosis: a prospective derivation and global validation study. Lancet Gastroenterol Hepatol. 2020;5(4):362–373. doi:10.1016/S2468-1253(19)30383-8 53. Xiao G, Zhu S, Xiao X, Yan L, Yang J, Wu G. Comparison of laboratory tests, ultrasound, or magnetic resonance elastography to detect fbrosis in patients with nonalcoholic fatty liver disease: a meta-analysis. Hepatology. 2017;66(5):1486–1501. doi:10.1002/hep.29302 54. Chen J, Yin M, Talwalkar JA, et al. Diagnostic performance of MR elastography and vibration-controlled transient elastography in the detection of hepatic fbrosis in patients with severe to morbid obesity. Radiology. 2017;283(2):418–428. doi:10.1148/radiol.2016160685 55. Bureau C, Metivier S, Peron JM, et al. Transient elastography accurately predicts presence of signifcant portal hypertension in patients with chronic liver disease. Aliment Pharmacol Ther. 2008;27(12):1261–1268. doi:10.1111/j.1365-2036.2008.03701.x 56. Bolognesi M, Merkel C, Sacerdoti D, Nava V, Gatta A. Role of spleen enlargement in cirrhosis with portal hypertension. Digestive and Liver Disease. 2002;34(2):144–150. doi:10.1016/S1590-8658(02)80246-8 57. Abraldes JG, Reverter E, Berzigotti A. Spleen stiffness: toward a noninvasive portal sphygmomanometer? Hepatology. 2013;57(3):1278–1280. doi:10.1002/hep.26239 58. Colecchia A, Montrone L, Scaioli E, et al. Measurement of spleen stiffness to evaluate portal hypertension and the presence of esophageal varices in patients with HCVrelated cirrhosis. Gastroenterology. 2012;143(3):646–654. doi:10.1053/j.gastro.2012.05.035 59. Franchis R de, Bosch J, Garcia-Tsao G, et al. Baveno VII—Renewing consensus in portal hypertension. J Hepatol. 2022;76(4):959–974. doi:10.1016/j. jhep.2021.12.022 60. Abraldes JG, Bureau C, Stefanescu H, et al. Noninvasive tools and risk of clinically signifcant portal hypertension and varices in compensated cirrhosis: the “Anticipate” study. Hepatology. 2016;64(6):2173–2184. doi:10.1002/hep.28824 61. Pons M, Augustin S, Scheiner B, et al. Noninvasive diagnosis of portal hypertension in patients with compensated advanced chronic liver disease. Off J Am Coll Gastroenterol | ACG. 2021;116(4):723–732. doi:10.14309/ ajg.0000000000000994

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8 MRI-Based Technologies Victor de Lédinghen CONTENTS 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 8.2 MRI for Steatosis Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 8.2.1 Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 8.2.2 MRI-PDFF for the Diagnosis of Steatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 8.2.3 Evaluation of Steatosis over Time with or without Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 8.2.4 Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 8.3 MRE for Liver Fibrosis Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 8.3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 8.3.2 MRE Technique and Image Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 8.3.3 MRE for the Diagnosis of Liver Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 8.3.4 Clinical Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 8.3.5 MRE for the Diagnosis of NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 8.3.6 Evaluation of Liver Fibrosis Overtime with or without Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 8.3.7 Comparison of Different Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 8.3.8 MRE for the Evaluation of the Severity of the Liver Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 8.3.8.1 Portal Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 8.3.8.2 Liver Events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 8.3.8.3 MRE and Cardiovascular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 8.3.9 MRE in Pediatric NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 8.4 3D MR Elastography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 8.5. Unresolved Issues to Be Addressed in the Future. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 8.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Key Points ■

MRI-PDFF is a useful surrogate for liver biopsy in the diagnosis of steatosis and assessment of treatment response.

■

MRE has several advantages over ultrasound-based elastography, as it samples a much larger volume of the liver, is not affected by body mass index or degree of steatosis, is not operator-dependent, has favorable testretest repeatability, and has a high success rate.

■

MRE is the most accurate noninvasive method for staging liver fbrosis.

■

MRE is an accurate prognostic noninvasive imaging biomarker that can risk-stratify patients with NAFLD.

8.1 INTRODUCTION Magnetic resonance imaging (MRI) is an imaging method with contrast mechanisms capable of enabling the detection and quantifcation of liver fat. Liver MR elastography (MRE) is an imaging technique used to measure liver stiffness in the evaluation for possible fbrosis or cirrhosis. In this chapter, we will mainly discuss MRI and MRE, which have revolutionized the noninvasive diagnosis of steatosis and liver fbrosis, respectively. However, we need to keep in mind that MRI cannot be performed in patients who have contraindications including metallic implants and severe claustrophobia. 8.2 MRI FOR STEATOSIS ASSESSMENT Quantitative imaging methods are increasingly used for the diagnosis and management of steatosis, including 74

treatment monitoring. Despite the high accuracy of MRI for detecting and grading steatosis, cost and limited availability restrict its use in clinical practice. 8.2.1 Technique MRI is an imaging method with contrast mechanisms capable of enabling the detection and quantifcation of liver fat content by means of the direct measurement of proton signal in water and fat (1). Magnetic resonance proton density fat fraction (MRI-PDFF) is an accurate, reproducible, quantitative imaging-based technique that has the ability to quantify liver fat in its entire dynamic range. MRI-PDFF is increasingly accepted as the most optimal method, among the invasive or noninvasive methods, for quantifying liver fat content—even overperforming biopsy. Currently, there is no consensus on a standardized approach to measuring liver fat with manually drawn regions of interest (ROIs). Because a heterogeneous pattern of steatosis has been reported in up to 60% of patients with NAFLD, the placement of a single ROI is unlikely to be suffcient to correctly estimate the true severity of liver fat. Although placement of largest-ft-possible ROIs in all liver segments was shown to be the most reproducible and repeatable method, it is time-consuming and thus diffcult for clinical practice. Therefore, placement of one large single-section ROI in the anterior, posterior, medial and lateral segments of the liver, avoiding bigger vessels and bile ducts, has been proposed as an acceptable alternative. 8.2.2 MRI-PDFF for the Diagnosis of Steatosis The utility of imaging methods used for the assessment of steatosis is crucial. Moreover, given the high prevalence of steatosis, it is a common incidental f nding at DOI: 10.1201/9781003386698-10

8 MRI-BASED TECHNOLOGIES

cross-sectional imaging. This provides a unique opportunity to report and grade the severity of steatosis to initiate lifestyle modifcations or other interventions. Table 8.1 reports some studies that evaluated the performance of MRI-PDFF for the diagnosis of steatosis. In a recent meta-analysis (2), the areas under the ROC curve (AUROCs) of MRI-PDFF for detecting steatosis >−5%, >−33% and >−66% were 0.98, 0.91 and 0.90, respectively. MRI-PDFF could be also a valuable biomarker for preoperative risk assessment in donor candidates for living donor liver transplant (3). At last, MRI-PDFF has high diagnostic accuracy to classify steatosis grade in histological steatosis grade in children with NAFLD (4). Practice guidance statements from the American Association for the Study of Liver Diseases recommend the following for incidentally detected steatosis at imaging: 1. Patients with abnormal liver function tests or signs attributable to liver disease should be evaluated as suspected for NAFLD and approached accordingly. 2. Patients with normal liver function tests should be assessed for metabolic risk factors such as obesity, dyslipidemia or diabetes mellitus or for other alternative causes for steatosis such as excessive alcohol consumption or possibly medication induced. In patients with abnormal liver function tests or incidental fndings of hepatic steatosis at imaging and high clinical suspicion for NAFLD, a rapid MRI protocol targeted for liver fat quantifcation is the method of choice for estimating the severity of steatosis. 8.2.3 Evaluation of Steatosis over Time with or without Treatment A major obstacle in NASH therapeutic trials is the need for repeated liver biopsies to assess the longitudinal treatment response, and these are invasive and subject to sampling error and variability in interpretation. In this context, MRI-PDFF has emerged as a noninvasive and reproducible alternative in assessing NASH and monitoring the treatment response, as well as in aiding the initial NASH diagnosis. MRI-PDFF is one of the leading imaging-based biomarkers of assessing antisteatotic benefts of a drug therapy in NASH. MRI-PDFF is frequently used as an endpoint in early-phase NASH trials. Because of its increased utilization in NASH clinical trials, there is a need for standardization of criteria for assessing treatment response in NASH trials. The optimal cut-point that was associated with histologic response was noted to be ≥30% relative reduction in MRI-PDFF. Subsequently, a recent metaanalysis has been published that provides pooled estimates on the association between MRI-PDFF responders, defned as ≥30% reduction in MRI-PDFF relative to baseline and histologic response. Seven studies were examined

in this meta-analysis, including 346 subjects. The rate of histologic response as defned as ≥2-point improvement in NAS in MRI-PDFF responders vs. nonresponders was 51 vs. 14% (P value < 0.01), respectively, and the rate of NASH resolution as defned as 0 ballooning and 0–1 in lobular infammation in MRI-PDFF responders vs. nonresponders was 41 vs. 7% (P value < 0.01), respectively (9). In a secondary analysis of a prospective phase Ib clinical trial evaluating a candidate treatment (MET409, a farnesoid X receptor agonist) for NASH, 48 participants were analyzed at baseline and at 4 and 12 weeks after active treatment with either MET409 (n=30) or placebo (n=18) treatment (10). An at least 19.3% relative MRI-PDFF reduction at W4 yielded an AUROC of 0.98 (sensitivity, 89%; specifcity, 95%) for predicting an at least 30% relative MRI-PDFF reduction at W12. Therefore, early MRI-PDFF measurements may serve as early indicators of the treatment effect, as early indicators of the treatment response, and as potential early endpoints in NASH trials. Finally, MRI-PDFF has high diagnostic accuracy to predict histological steatosis change in histological steatosis grade in children with NAFLD (4). In conclusion, MRI-PDFF responder is defned as a ≥30% relative reduction in MRI-PDFF between baseline and end of treatment. Super-responder on MRI-PDFF is defned as a ≥50% relative reduction in MRI-PDFF between baseline and end of treatment. This is associated with signifcantly higher rates of NASH resolution. However, MRI-PDFF may not be useful for therapeutic agents that target primarily either infammation or fbrosis and do not have any metabolic effects. 8.2.4 Guidelines Recently, EASL published its guidelines about noninvasive tests for the evaluation of liver disease severity and prognosis (11). First, they indicated that noninvasive scores are not recommended for the diagnosis of steatosis in clinical practice (LoE 2; strong recommendation). But they added that MRI-PDFF is the most accurate noninvasive method for detecting and quantifying steatosis. However, it is not recommended as a frst-line tool given its cost and limited availability. Therefore, it is more suited to clinical trials (LoE 2; strong recommendation). MRI-PDFF can be used to assess steatosis evolution under treatment (LoE 2; weak recommendation). However, the minimal decrease in MRIPDFF that defnes a clinically relevant change or treatment response needs to be better def ned. 8.3 MRE FOR LIVER FIBROSIS ASSESSMENT 8.3.1 Introduction Elastography is an imaging technique used to evaluate the mechanical properties of tissue according to the propagation of mechanical waves. MRI is coupled with a device

Table 8.1: Diagnosis of Steatosis Using MRI-PDFF Author

Year

N

Gu J (2) Middleton MS (4) Park CC (5) Imajo K(6) Tang A (7) Idilman I (8)

2019 2018 2017 2016 2013 2013

635 meta-analysis 110 Children 104 142 77 70

Steatosis Grades 2 and 3

Steatosis Grade 3

0.91 0.87 (0.80, 0.94) 0.90 (0.82–0.97) 0.90 (0.82–0.97 0.825 (0.734, 0.915) 0.95 (0.91, 1.00)

0.90 0.79 (0.70, 0.87) 0.92 (0.84–0.99) 0.893 (0.809, 0.977)

75

SECTION II: DIAGNOSTIC TESTS

that generates mechanical waves, typically shear waves within the tissue(s) of interest. The shear wave velocity is then measured to calculate quantitative results. The shear wave velocity in tissue is directly related to the stiffness of the tissue. Propagation of shear waves is faster in stiff or hard tissues and slower in soft tissues. Liver MRE is an imaging technique used to measure liver stiffness in the evaluation for possible fbrosis or cirrhosis. MRE has several advantages over ultrasound-based elastography, as it samples a much larger volume of the liver, is not affected by body mass index or degree of steatosis, is not operator dependent, has favorable test-retest repeatability, and has a high success rate (Table 8.2). 8.3.2 MRE Technique and Image Interpretation MRE estimates liver stiffness, which correlates to the amount of collagen deposition in the extracellular matrix, but liver stiffness is also infuenced by other factors, such as infammation, vascular congestion, and cholestasis. Consequently, in early NASH when infammation and cellular injury prevail over mild fbrosis, conventional MRE detects increased liver stiffness but cannot distinguish whether the increase is due to viscoelastic changes of infammation or due to mild fbrosis. In a typical liver MRE confguration, an active pneumatic mechanical wave driver is located outside the MRE room and is connected to a passive driver that is fastened onto the abdominal wall over the liver. The passive driver generates a continuous acoustic vibration that is transmitted through the entire abdomen, including the liver, at a fxed frequency, which is typically 60 Hz. The most commonly used clinical MRE pulse sequence approved by the US Food and Drug Administration is a two-dimensional gradient-recalled-echo MR elastography sequence. After the magnitude and phase images are created, an inversion algorithm installed in the MRI unit

automatically processes these raw data images to create several additional images and maps. The most common output images generated by MRI units are a color wave image depicting the propagation of shear waves through the abdomen, a grayscale elastogram without a superimposed 95% confdence map, a grayscale elastogram with a superimposed 95% confdence map, a color elastogram without a superimposed 95% confdence map, and a color elastogram with a superimposed 95% confdence map. The confdence map is a statistical derivation used to overlay a “checkerboard” on the stiffness map to exclude regions in the liver that have less reliable (i.e., noisy and discontinuous) stiffness data, so that a high-quality liver stiffness measurement can be obtained. The grayscale elastogram is commonly used to obtain quantitative liver stiffness measurements in kilopascals (kPa). The color elastogram is generally used for qualitative liver stiffness evaluation. However, the color elastograms created by MRI units from some vendors can also be used to obtain quantitative measurements. The color elastogram used clinically has a stiffness range of 0–8 kPa. Liver stiffness measurement are obtained in the largest measurable portion of the liver on each of the four elastograms. On each image, a mean liver stiffness measurement, in kilopascals, along with the ROI size, in square centimeters (cm2), is obtained. Then the overall mean liver stiffness is obtained by calculating the weighted arithmetic mean, which refects the relative contribution of the area of the liver measured on each image. On the magnitude images, which provide the best anatomic detail of the liver, it is important to avoid the liver edge (≥1 cm from liver edge), nonhepatic tissues, fssures, gallbladder fossa, and large blood vessels. The left hepatic lobe can have a signifcant motion artifact due to cardiac pulsations and thus should be avoided as well, unless no motion artifact is identifed. On the wave images, areas

Table 8.2: Comparison of Elastography Techniques (11)  

Transient Elastography

pSWE

2D-SWE

MRE

Can be performed in combination with regular ultrasound if the device is provided with adequate software Large ROI that can be adjusted in size and location chosen by the operator Measures liver stiffness in real time Good applicability High performance for the diagnosis of signifcant fbrosis and cirrhosis Prognostic value in compensated cirrhosis No clear cutoffs for the diagnosis of advanced fbrosis or cirrhosis No strong evaluation of liver fbrosis evolution over time

Can be implemented on a regular MRI machine Examination of the whole liver Higher applicability than TE (ascites, obesity) High performance for the earlier fbrosis stage and for diagnosis of cirrhosis Used in clinical trials for the assessment of fbrosis regression

Advantages

Most widely used and validated technique Point-of-care (bedside, rapid, easy to learn) Quality criteria well-defned Good reproducibility High performance for cirrhosis (AUROC >0.9) Prognostic value in compensated cirrhosis well-validated

Can be performed in combination with regular ultrasound if the device is provided with adequate software ROI smaller than TE and location chosen by the operator Higher applicability than TE (ascites and obesity) Performance equivalent to that of TE and advanced fbrosis and cirrhosis High applicability for spleen stiffness measurement

Disadvantages

Requires a dedicated device ROI cannot be chosen

No clear cutoffs for the diagnosis of advanced fbrosis or cirrhosis No strong evaluation of liver fbrosis evolution over time

Requires an MRI facility Time-consuming Costly No strong data on prognostic value

2D-SWE: Bidimensional shear wave elastography; MRE: Magnetic resonance elastography; MRI: Magnetic resonance imaging; pSWE: Point-shear wave elastography; ROI: Region of interest; TE: Transient elastography.

76

8 MRI-BASED TECHNOLOGIES

of poor wave propagation, wave distortion and lowamplitude waves should be avoided. On the grayscale and color elastograms, the crosshatched regions on the superimposed 95% confdence map must be excluded from measurements. Finally, on the color elastogram, hot spots need to be recognized and excluded from measurements. Obtaining and reporting accurate and reliable liver stiffness measurements with MRE requires an understanding of the three core components of liver MRE: optimization of imaging technique, prompt quality control of images, and proper interpretation and reporting of elastogram fndings. When performing MRE, six technical parameters should be optimized: ■

Patient fasting before the examination

■

Proper passive driver placement

■

Proper MRE section positioning over the largest area of the liver

■

Use of MRE-related sequences at end expiration

■

Choosing the best timing of the MRE sequence

■

Optimization of several essential pulse sequence parameters

respectively (14). Loomba et al. proposed that a MRE liver stiffness cutoff of 4.67 kPa could predict cirrhosis. In another study from the same center, Park et al. reported that an MRE liver stiffness cutoff of 3.35 kPa could detect cirrhosis. Cui et al. found that a cutoff of 4.15 kPa distinguished cirrhosis from other stages. Imajo et al. found that a slightly higher MRE liver stiffness cutoff 6.7 kPa detected cirrhosis (19). In a recent study, MRE liver stiffness of 4.7 and 5.5 kPa was suggested as detecting advanced fbrosis and cirrhosis, respectively. A pooled analysis of 230 NAFLD patients suggested that the optimal cutoff for cirrhosis was 4.7 kPa. At last, Hsu et al. proposed 3.62 kPa and 4.7 kPa for the diagnosis of advanced fbrosis and cirrhosis, respectively (20). At this time, there is no guidelines for any cutoff value for the diagnosis of advanced fbrosis and cirrhosis. 8.3.5 MRE for the Diagnosis of NASH Liver biopsy remains the reference standard for the diagnosis of NASH because none of the available noninvasive tests has acceptable accuracy (LoE 2) (11). But, recently, a meta-analysis of 224 patients (4 studies) reported an AUROC of 0.83 (0.69–0.91) for the diagnosis of NASH using MRE with a sensitivity of 0.65 (0.46–0.80), and a specifcity of 0.83 (0.69–0.91) (12). Loomba et al. suggested that a cutoff value of 3.26 kPa could discriminate NASH from NAFL, which was similar to another study by Costa-Silva et al., who reported a cutoff of 3.2 kPa (21) (13).

As soon as the MRE examination is performed, the elastograms should be reviewed to ensure that they are of diagnostic quality so that corrective steps can be taken, if needed, and the MRE can be repeated before the diagnostic portion of the examination concludes. 8.3.3 MRE for the Diagnosis of Liver Fibrosis Several studies demonstrated the value of MRE for a noninvasive detection of liver fbrosis and the potential of MRE for distinguishing different fbrosis stages in NAFLD. The main results of different studies are indicated in Table 8.3. 8.3.4 Clinical Interpretation As with all diagnostic studies, the choice of a cutoff value for the diagnosis of advanced fbrosis or cirrhosis is crucial. The choice of this value depends on the practitioner’s wish: preference for better sensitivity or better specifcity? In a meta-analysis including 9 studies and 232 patients with NAFLD, liver stiffness measurement cutoffs of 3.77 and 4.09 kPa discriminated advanced fbrosis and cirrhosis, respectively (18). In another recent meta-analysis, the cutoff values of 3.62–4.8 kPa and 4.15–6.7 kPa were proposed for the diagnosis of advanced fbrosis and cirrhosis,

8.3.6 Evaluation of Liver Fibrosis Overtime with or without Treatment In NAFLD, the severity of histological features, such as steatohepatitis without fbrosis, does not predict the prognosis of patients, whereas only fbrosis predicts a long-term prognosis (22). There are limited longitudinal data on the association between changes in MRE and liver fbrosis on paired liver biopsies. A prospective cohort study included 102 patients with biopsy-proven NAFLD who underwent contemporaneous MRE and liver biopsy at baseline followed by a repeat paired liver biopsy and MRE assessment (23). The median time interval between the two paired assessments was 1.4 years. In unadjusted analysis, a 15% increase in MRE was associated with increased odds of histologic fbrosis progression (OR 3.56; 95% CI, 1.17–10.76; P = 0.0248). These fndings remained clinically and statistically signifcant even after multivariable adjustment for age, sex and BMI (adjusted OR, 3.36; 95% CI, 1.10–10.31; P = 0.0339). A 15%

Table 8.3: Performance of MRE for the Diagnosis of Liver Fibrosis in NAFLD  

Year

N

AUROC F2F3F4 (95% CI)

AUROC F3F4 (95% CI)

AUROC F4 (95% CI)

Selvaraj E. A. (12) Costa Silva L. (13) Xiao G. (14) Park C. C. (5) Imajo K. (6) Cui J. (15) Kim D. (16) Lomba R. (17)

2021 2018 2017 2017 2016 2016 2014 2014

Meta-analysis 14,609 49 Meta-analysis 384 94 142 125 142 117

0.91 (0.80–0.97) 0.932 (0.823–0.984) 0.92 0.89 (0.85–0.96) 0.89 (0.85–0.94) 0.885 (0.816–0.953)

0.92 (0.88–0.95) 0.928 (0.817–0.982) 0.96 0.87 (0.78–0.96) 0.89 (0.83–0.95) 0.934 (0.863–1.000) 0.954 (0.905, 0.982) 0.924

0.90 (0.81–0.95) 0.964 (0.867–0.996) 0.97 0.87 (0.71–1.00) 0.97 (0.94–1.00) 0.882 (0.729–1.000)

0.856

0.894

AUROC: Area under the ROC curve; CI: Confdence interval.

77

SECTION II: DIAGNOSTIC TESTS

increase in MRE was the strongest predictor of progression to advanced fbrosis (OR, 4.90; 95% CI, 1.35–17.84; P = 0.0159). Therefore, a 15% increase in liver stiffness on MRE may be associated with histologic fbrosis progression and progression from early fbrosis to advanced fbrosis (23). However, these results should be confrmed in ongoing clinical trials. 8.3.7 Comparison of Different Methods MRE and transient elastography (FibroScan®) have been compared head to head in patients with biopsy-proven NAFLD in a small number of studies. These studies found that MRE is equal or more accurate than FibroScan® in identifcation of liver fbrosis, using liver biopsy analysis as the standard (Table 8.4). A study evaluated the performances of MRE vs. ARFI for diagnosing fbrosis in NAFLD patients (15). For diagnosing any fbrosis (>stage 1), the MRE AUROC was 0.799 (95% CI 0.723–0.875), signifcantly higher than the ARFI AUROC of 0.664 (95% CI 0.568–0.760). In a recent metaanalysis, it was shown that MRE and shear wave elastography have the highest diagnostic accuracy for staging fbrosis in NAFLD patients (12). The American Gastroenterological Association published clinical guidelines on the role of FibroScan® and MRE for the diagnosis of liver fibrosis in NAFLD (25). They conclude that MRE has little to no increased diagnostic accuracy in identifying cirrhosis in patients who truly have cirrhosis over FibroScan® but has considerably higher diagnostic accuracy in ruling out cirrhosis in patients who do not have cirrhosis, over FibroScan® (very low quality of evidence). EASL guidelines conclude that MRE is the most accurate noninvasive method for staging liver fibrosis (11). However, it is only marginally better than other noninvasive tests for F3–F4 fibrosis, and it is not recommended as a first-line given its cost and limited availability (LoE 2; strong recommendation). Therefore, it is more suited to clinical trials. AASLD guidelines conclude that FibroScan® and MRE are clinically useful tools for identifying advanced fbrosis in patients with NAFLD (26). At last, French guidelines indicate that the measurement of liver stiffness by MR could be useful (27).

8.3.8 MRE for the Evaluation of the Severity of the Liver Disease 8.3.8.1 Portal Hypertension That liver stiffness correlates with the degree of portal hypertension has been shown in many studies using FibroScan® or ultrasound devices. It is also the case with MRE. Recently, 52 patients with cirrhosis underwent hepatic vein catheterization and 2D-MRE on separate days (28). Thirty-six of the patients had a hepatic venous pressure gradient (HVPG) of ≥12 mmHg and were tested prior to and after intravenous infusion of nonselective beta-blockers (NSBB) using HVPG measurement and MRE. HVPG showed a strong, positive, linear relationship with liver stiffness (r2 = 0.92; P < 0.001) and spleen stiffness (r2 = 0.94; P < 0.001). The cutoff points for identifying patients with a HVPG ≥ 12 mmHg were 7.7 kPa for liver stiffness (sensitivity, 0.78; specifcity, 0.64) and 10.5 kPa for spleen stiffness (sensitivity, 0.8; specifcity, 0.79). Intravenous administration of NSBB signifcantly decreased spleen stiffness by 6.9% (CI: 3.5–10.4, P < 0.001), but NSBB had no consistent effect on liver stiffness. However, changes in spleen stiffness were not related to the HVPG response (P = 0.75). An estimation by 2D-MRE of liver or spleen stiffness could refect the degree of portal hypertension in patients with liver cirrhosis, but changes in stiffness after NSBB do not predict the effect on HVPG. 8.3.8.2 Liver Events Fibrosis stage on histology has been shown to be a strong predictor of liver-associated outcomes (29). Liver stiffness measurement with FibroScan® is associated with liver events and prognosis in chronic liver diseases, especially NAFLD (30,31). The same results were published about liver stiffness measurement with MRE. A recent retrospective cohort study of 829 adults with NAFLD who underwent MRE showed that baseline liver stiffness measurement was predictive of future cirrhosis development (32). Moreover, baseline liver stiffness measurement was predictive of future decompensation or death. The 1-year probability of future decompensation or death in cirrhosis with baseline LSM of 5 kPa vs. 8 kPa was 9% vs. 20%, respectively.

Table 8.4: Comparison of Different Methods for the Diagnosis of Advanced Liver Fibrosis and Cirrhosis Author

Year

N

AUROC MRE

AUROC TE

AUROC 2D-SWE

AUROC MRE

Advanced fbrosis Sevaraj E. A. (12) Imajo K. (19) Furlan A. (24) Hsu C. (20) Park C. C. (5)

2021 14,609 0.92 meta-analysis (0.88–0.95) 2020 231 0.937 (0.882–0.958) 2019 59 0.95 (0.89–1.00) 2019 230 0.93 (0.89–0.96) 2017 104 0.87 (0.78–0.96)

0.85 0.72 (0.83–0.87) (0.60–0.84) 0.924 0.920 (0.867–0.947) (0.865–0.953) 0.86 0.89 (0.77–0.95) (0.80–0.98) 0.84 (0.78–0.90) 0.80 (0.67–0.93)

AUROC TE

AUROC 2D-SWE

p

Cirrhosis 0.90 0.89 0.88 (0.81–0.95) (0.84–0.93) (0.81–0.91) 0.923 0.872 0.886 5 being a risk factor. The prevalence of steatosis increased with OSA severity with an OR of 2.33 for an AHI between 5 and 30 and an OR of 2.80 for AHI >30 events per hour.42 However, there was no association between AHI and fbrosis in 185 subjects.42 In contrast, a prospective observational study of 51 nonbariatric patients with biopsy-proven NAFLD, those with moderate to severe OSA had a slightly increased risk of hepatic fbrosis (OR 1.22).43 Therefore, while some conficting data exist, most studies in adults show a strong relationship between OSA and NAFLD disease severity. However, little is known about the impact of chronic intermittent hypoxia on NAFLD disease severity. Small pediatric studies have provided some insight into the role of OSA/CIH on NAFLD disease severity in children. Early-onset NAFLD is associated with an increased risk of complications later in life, including cirrhosis and hepatocellular carcinoma.44 Sundaram et al. frst demonstrated in obese children with biopsy-proven NAFLD that OSA/CIH was common, occurring in 60% of the studied subjects. They focused on the impact of CIH, noting that children with OSA/CIH had signifcantly more severe histologic hepatic fbrosis, which was related to a worsening oxygen nadir. In addition, increasing time with oxygen saturations below 90% was related to NAFLD histologic infammation, steatosis and NAS score, as well as aminotransferase elevation.45 They went on to further show that nocturnal hypoxia was a trigger for localized oxidative stress, an important factor associated with NASH progression and hepatic fbrosis in children.32 They also demonstrated that mechanistically, the hedgehog pathway was activated in pediatric patients with NAFLD and nocturnal hypoxia, which again related to disease severity.39 Nobili et al. also studied children with biopsy-proven NAFLD, noting that the duration of oxygen desaturations correlated with increased intrahepatic leukocytes and activated macrophages (Kupffer cells), circulating markers of hepatocyte apoptosis (CK-18) and hepatic fbrogenesis (hyaluronic acid).46 A recent meta-analysis by Chen et al. aimed to evaluate the relationship between OSA and NAFLD in children and adolescents based on 9 pediatric studies. Both AST and ALT were signifcantly higher in children with OSA than control groups. Subgroup analyses revealed that both mild and severe OSA were signifcantly correlated to elevated liver enzymes. Focusing on the 2 studies that evaluated NAFLD histologically, they found NAFLD infammation tended to be higher in OSA patients and that OSA was signifcantly associated with worse hepatic fbrosis.47 TREATMENT Continuous positive airway pressure (CPAP) is the gold standard treatment for OSA. It precludes upper airway collapse, thereby improving sleep fragmentation, symptoms such as daytime sleepiness, and overall quality of life.48,49 Importantly, in patients with OSA, CPAP treatment decreases mortality risk.50 Given the emerging relationship between OSA and NAFLD, CPAP treatment for NAFLD 170

is an intriguing concept. To date, published studies have yielded conficting results for several reasons. First, NAFLD and NASH have been variably defned in the published studies. Histologic confrmation and determination of NAFLD severity by the gold standard of liver biopsy is a rarity in this segment of the published literature. Most commonly, abnormal aminotransferases have been used to defne patients as having NAFLD. Additional criteria used to defne NAFLD in these studies include the presence of steatosis on radiologic imaging (ultrasound, CT scan or MRI), as well abnormal controlled attenuation parameter (CAP) on FibroScan® testing. Post-CPAP outcomes measures have also been variable, including the markers used to determine outcomes. Moreover, individual studies have had relatively small sample sizes. The length of CPAP treatment has also been quite variable, with few studies extending beyond 12 months, as would be common in pharmacologic trials. Finally, details regarding adherence to CPAP therapy are scarce and likely further impact the interpretation of studies’ results. As such, each study must be interpreted in the context of these potential shortcomings. CPAP therapy has most extensively been studied to determine its effects on aminotransferases in NAFLD. An observational study utilized nasal CPAP, demonstrating decreased AST after 1 month, with sustained improvements over 6 months of CPAP therapy.51 A very small prospective cohort study of 6 subjects treated with CPAP for 3 years demonstrated signifcant reduction in liver enzymes as compared to 5 controls, without changes in BMI confounding these results.52 A study of 28 patients with ultrasound-defned NAFLD had signifcant improvements in both AST and ALT, without any change in BMI, after 3 months of CPAP (mean usage: 6 hours per day).53 More recently, an observational study utilizing an institutional database of 351 subjects receiving CPAP for at least 3 months found signifcant improvement in liver enzymes.54 This study suggested that those with better adherence had greater decreases in their aminotransferases.54 Sundaram et al. studied adolescents with biopsy-proven NAFLD, severe OSA/CIH and evidence of signifcant oxidative stress. Despite an increase in body mass index, 3 months of home CPAP therapy reduced ALT, metabolic syndrome markers and oxidative stress, as measured by F(2)-isoprostanes.55 Kohler et al. conducted a 4-week randomized control trial of patients with NAFLD based on elevated liver enzymes. In this study, no benefcial effect on aminotransferases in the 47 subjects receiving CPAP was found as compared to 47 controls who received subtherapeutic CPAP.56 Using a slightly different approach, Sivam et al. studied 27 subjects in a randomized control crossover trial. They all received CPAP for 8 weeks, with no changes in aminotransferase levels detected.57 A recent metaanalysis examined the clinical utility of CPAP treatment for NAFLD, which included 5 articles with 7 patient cohorts and 192 adult subjects. This meta-analysis showed that CPAP use was associated with signifcant decreases in both AST and ALT in patients with OSA, without concurrent changes in BMI. Importantly, subgroup analyses indicated that 3 or more months of CPAP treatment were required to garner this clinical beneft.53 As such, while non-RCTs and a meta-analysis suggest CPAP therapy may be benefcial in improving aminotransferases in NAFLD, small RCTs do not support this treatment. The relatively short duration of therapy in these RCTs may have these negative results. CPAP has also been evaluated for its utility in treating the hepatic steatosis central to NAFLD physiology. In

18 OBSTRUCTIVE SLEEP APNEA AND NONALCOHOLIC FATTY LIVER DISEASE

their small cohort study, Shpirer et al. found that 2–3 years of CPAP partially reversed moderate to severe steatosis assessed by CT scan, with no accompanying changes in the BMI or triglyceride levels of participants.52,58 In an observational cohort study, 6–12 months of CPAP improved severe steatosis without changes in BMI.59 Toyama et al. conducted an observational study of 61 subjects. In those with hepatic steatosis at baseline, there was decreased accumulation of liver fat after 31 months of CPAP therapy, despite stable BMI and worsening visceral and subcutaneous fat accumulations, suggesting longer therapy may be needed to alter hepatic steatosis.60 In 50 subjects with OSA, hepatic steatosis was assessed by CAP score using FibroScan®. After 6 months of CPAP, CAP scores for steatosis did not improve. Furthermore, a subgroup analysis of 17 subjects with stable BMI and good CPAP adherence also did not show improved CAP measurements.61 In contrast, the previously mentioned RCT with crossover design of 8 weeks of CPAP therapy did not improve hepatic fat as measured by magnetic resonance imaging and spectroscopy. Kritikou et al. also found no improvement in hepatic steatosis by CT scan in an RCT of 38 subjects receiving either CPAP or sham CPAP for 2 months.62 Similarly, an RCT of 65 subjects found no signifcant change in liver fat after 24 weeks of either CPAP or sham CPAP.63 Most recently, an RCT of 106 subjects with clinical NAFLD (criteria not defned) and OSA (based on a home sleep test, not polysomnography) received 6 months of either autoadjusting or subtherapeutic CPAP. Again, no differences in hepatic fat measured by MR spectroscopy and controlled attenuation parameter (CAP) scores by transient elastography were found, even when only looking at subjects with at least 4 hours of CPAP usage per night.64 CPAP treatment for hepatic steatosis thereby yields results similar to outcomes with liver enzymes; observational studies suggest the potential beneft of CPAP while RCTs do not.

Limited data are available on the impact of CPAP for NAFLD hepatic fbrosis treatment, although fbrosis may be the most important indicator of long-term prognosis. Kim et al. used an institutional database of 221 subjects with OSA and clinically suspected NAFLD based on elevated ALT. In subjects treated with CPAP for at least 3 months, a potential reduction in advanced fbrosis as assessed by APRI (AST-to-platelet ratio index) score, a surrogate for fbrosis, was seen.54,65 Liver fbrosis based on serum lysyl oxidase, which cross-links collagen and may be a biomarker of fbrosis, was also signifcantly reduced in 35 subjects after 3 months of CPAP therapy.66 However, 50 subjects with OSA and NAFLD, demonstrated no changes in liver stiffness measurements indicating fbrosis by FibroScan®.61 Similarly, Buttacavoli studied 15 patients before and after 6–12 months of CPAP and found no improvement in fbrosis by transient elastography.59 An RCT to assesses liver fbrosis improvement using CPAP vs. sham treated 103 patients for 6–12 weeks without improvements in Fibromax scores.67 FibroMax is a proprietary noninvasive test that uses an algorithm that includes gender, age, weight and height with 10 serum biomarkers. FibroMax includes three tests, SteatoTest, NashTest and FibroTest, in order to evaluate steatosis, NASH and liver fbrosis, respectively.67 Most recently, an RCT of 106 subjects with clinical NAFLD received 6 months of either autoadjusting or subtherapeutic CPAP with no improvements in liver stiffness as measured by transient elastography, even when focusing only on the subgroup with at least 4 hours of CPAP usage per night.64 The clinical beneft of screening every patient with NAFLD for OSA and vice versa is unclear. However, simple clinical screening tools in clinic and liver function tests could help determine who needs further assessment or evaluation. As the availability of noninvasive point of

Table 18.1: Studies Examining the Therapeutic Benefit of CPAP on NAFLD Author

Study Design

CPAP Duration

Outcome of interest

Therapeutic Beneft

Chin51 Shpirer 52

Observational Prospective Cohort

40 11 (6 CPAP, 5 control)

Sample Size 

6 months 3 years

Yes Yes and yes

Chen68 Kim54

Prospective Cohort Observational

160 351

3 months 3 months

Sundaram55

Prospective Cohort

12

3 months

Kohler56 Sivam57 Chen53 Buttacavoli59 Toyama60 Hirono61

RCT* RCT*, cross over Meta-analysis Observational Observational Prospective cohort

94 (47 CPAP, 47 Sham) 27 192 15 61 123

1 month 8 weeks ≥ 3months 6–12 months 32 months 6 months

Kritikou62 Hoyos63 Ng64

RCT* Prospective cohort RCT*

2 months 3–6 months 6

Mesarwi66 Jullian-Desayes67

Prospective cohort RCT*

38 65 106 (53 CPAP, 53 subtherapeutic CPAP) 35 103 (51 CPAP, 52 sham)

Aminotransferases Aminotransferases and steatosis Aminotransferases Aminotransferases and fbrosis Aminotransferases and oxidative stress Aminotransferases Aminotransferases Aminotransferases Steatosis and Fibrosis Steatosis Aminotransferases and Steatosis Steatosis Steatosis Steatosis and fbrosis

3 months 1.5–3 months

Fibrosis Steatosis and fbrosis

Yes No and No

Yes Yes and yes Yes and yes No No Yes Yes and no Yes Yes and No No No No and no

RCT: Randomized control trial.

171

SECTION IV: EXTRAHEPATIC MANIFESTATIONS

Figure 18.1 Impact of obstructive sleep apnea and chronic intermittent hypoxia on NAFLD pathogenesis13

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19 EXTRAHEPATIC GASTROINTESTINAL MANIFESTATIONS OF NONALCOHOLIC FATTY LIVER DISEASE

19 Extrahepatic Gastrointestinal Manifestations of Nonalcoholic Fatty Liver Disease Rinjal Brahmbhatt and Mousab Tabbaa

CONTENTS 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 19.2 NAFLD and the Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 19.2.1 Gastroesophageal Refux Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 19.2.2 Barrett’s Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.2.3 Esophageal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.3 NAFLD and the Stomach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.3.1 Helicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.3.2 Gastric Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.4 NAFLD and the Small Intestines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.4.1 Celiac Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.4.1.1 Small Intestinal Bacterial Overgrowth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176 19.4.2 Infammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 19.5 NAFLD and the Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 19.5.1 Irritable Bowel Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 19.5.2 Diverticular Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 19.5.3 Clostridioides diffcile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 19.5.4 Colonic Adenomas and Colorectal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 19.6 NAFLD and the Pancreaticobiliary Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 19.6.1 Biliary Disease and Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 19.6.2 Pancreatitis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 19.6.3 Pancreatic Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 19.7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 Key Points ■

Nonalcoholic fatty liver disease (NAFLD) is a manifestation of metabolic disturbances and pathophysiologic pathways that impact all organ systems, and the gastrointestinal tract is no exception.

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Esophageal, gastric, small bowel, colonic and pancreaticobiliary diseases are prevalent in this population. This has important implications in the management of NAFLD as a multisystem disease.

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Of note, NAFLD is associated with several GI malignancies, and with earlier recognition and diagnosis, this may alter their disease course.

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In this chapter, we will review the impact of NAFLD on the gastrointestinal tract outside the parenchymal liver.

19.1 INTRODUCTION NAFLD is a multisystem disease associated with a myriad of extrahepatic gastrointestinal manifestations. Visceral adiposity and hepatic steatosis promote a state of systemic infammation, predisposing individuals with NAFLD to a variety of gastrointestinal diseases and extrahepatic neoplasias. Different mechanisms for this association have been proposed, including activation of the infammatory pathway by lipotoxicity that results in extrahepatic tissue damage. Herein, we describe the extrahepatic gastrointestinal manifestations of NAFLD and highlight studies that provide mechanistic explanations for these manifestations, as well as identify potentially modifable or clinically detectable relationships between NAFLD and these associated conditions. DOI: 10.1201/9781003386698-23

19.2 NAFLD AND THE ESOPHAGUS 19.2.1 Gastroesophageal Refux Disease Multiple studies have shown that NAFLD is independently associated with an increased risk of gastroesophageal refux disease (GERD) symptoms as well as erosive esophagitis (EE). The detrimental effect of NAFLD on EE might be greater than those of generalized and visceral obesity (1–3). Specifcally, Mikolasevic et al. demonstrated a positive association between controlled attenuation parameter (CAP) on transient elastography and a higher prevalence of GERD. In particular, they found a higher prevalence of LA grades B and C erosive esophagitis in this cohort of NAFLD patients (4). Interestingly, GERD may not only be associated with NAFLD but also may worsen it, representing a viscous cycle. Taketani et al. reported that nearly 30% of Japanese patients with biopsy-proven NAFLD had insomnia, which was independently associated with GERD symptoms (5). They also demonstrated that treatment with a proton pump inhibitor could relieve both insomnia and GERD symptoms. Correspondingly, Spiegel et al. demonstrated that a shorter sleep duration may increase the risk of obesity and diabetes due to changes in the secretion of hormones such as cortisol, leptin and ghrelin and to increased insulin resistance (6). Several plausible mechanisms to explain the relationship between NAFLD and GERD have been proposed. Increased serum levels of interleukin-1 and interleukin-6 in patients with NAFLD have been found to contribute to the development of GERD. Interleukin-6 could decrease esophageal contraction, impair acid clearance and thus increase refux episodes. Furthermore, increased systemic oxidative stress and impaired antioxidant capacity in

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patients with NAFLD may contribute to esophageal mucosal injuries, suggesting the critical role of oxidative stress in the pathogenesis of GERD. Finally, obesity, especially visceral adiposity in NAFLD patients, might increase intra-abdominal pressure leading to increased lower esophageal sphincter relaxation and therefore acid refux. Similarly, gastroparesis in NAFLD patients with diabetes may also increase the propensity for GERD. 19.2.2 Barrett’s Esophagus Obesity has been a well-established risk factor for Barrett’s esophagus (BE). Multiple studies have demonstrated an association between increased visceral adiposity and an increase in the risk of BE. In one study of a Japanese population with NAFLD, obesity tended to be associated with the risk of BE, and this risk appeared to be mediated for the most part by visceral adiposity (7–10). Apfel et al. reported an increased risk of progression of BE with low-grade dysplasia in one population of patients with cirrhosis, the majority of which had NASH as the etiologic cause of their liver disease. Furthermore, due to this association, the authors recommended increased surveillance of BE in such patients and, in particular, those with higher Child–Pugh scores (11). 19.2.3 Esophageal Cancer Esophageal cancer is the eighth most common form of cancer worldwide, and obesity has been identifed as a major risk factor. The risk of esophageal cancer has been found to be up to 4-fold higher in patients with obesity compared to lean populations and is even more pronounced in those with visceral fat distribution as compared to those with an increased BMI (12). Interestingly, the association between visceral obesity and esophageal adenocarcinoma is independent of GERD and possibly mediated by adipose tissue insulin resistance and chronic infammation (13–15). Purported mechanisms include upregulation of GGT, which is increased after oxidative stress. Previous studies have reported on the associations of serum GGT level with the risk of cancer. As essential parts of the cellular defense apparatus, GGT and GSH combat oxidative stress (16). Increased GGT has been regarded as a marker of exposure to certain carcinogens and its levels can be affected by environmental and lifestyle factors such as diet, tobacco use and alcohol use. Importantly, compared to patients without NAFLD, patients with NAFLD with incidental esophageal, stomach or colorectal cancer showed signifcantly increased all-cause mortality during the observation period in one study. Given the increased mortality in cancer patients with NAFLD, adipocytokines might link obesity-related disorders with neoplasm development both intra- and extrahepatically. The steatotic and infamed liver may secrete growth factors, such as NAFLD-derived plasminogen activator inhibitor 1, vascular endothelial growth factor (VEGF), or angiopoietin, into the systemic circulation and may therefore be involved in metastasis and cancer progression (17). 19.3 NAFLD AND THE STOMACH 19.3.1 Helicobacter pylori The possible association between Helicobacter pylori infection and NAFLD initially stemmed from the isolation of Helicobacter pylori (HP) in the livers of patients with NAFLD. Although there have been conficting results, several subsequent clinical trials have demonstrated a higher 176

rate of fatty liver and NASH in HP-positive patients compared to HP-negative patients. In one meta-analysis, the pooled overall odds ratio of H. pylori infection in NAFLD patients compared with controls was 1.36 (95% CI: 1.22– 1.53, I = 89.6%, P=0.000), and there was a 36% increased risk of NAFLD in patients with HP infection (18). Additionally, small trials examining the effect of HP eradication have shown improvement in markers of NAFLD activity, further supporting a link between these two conditions (19). Specifcally, treatment of HP resulted in an improved metabolic profle as defned by insulin resistance, the fatty liver index and imaging characteristics after HP was eradicated (20). Mechanisms include metabolic insults from infammatory cytokines such as CRP, TNF-α and IL-6, which trigger insulin resistance (21). Furthermore, HP may infuence leptin and adiponectin production, thereby infuencing fat metabolism and transporting relevant enzymes or by insulin signal transduction. Previous studies have also shown that the existence of HP itself was associated with increased intestinal permeability and may affect the normal gut microbiota, which is associated with metabolism and gut infammation. Some speculate that HP may invade the intestinal mucosa, increasing gut permeability and gut dysbiosis, thus facilitating the passage of bacterial endotoxin, mainly lipopolysaccharide, via the portal vein to the liver where it promotes the infammatory response leading to NAFLD (22) (Figure 19.1). 19.3.2 Gastric Cancer A possible direct link between NAFLD and gastric cancer has been suggested in a few studies (23). In one Turkish study, 1,840 patients underwent upper endoscopies over a 6-month period, and the prevalence of NAFLD in subjects with gastric cancer was higher than average (24). 19.4 NAFLD AND THE SMALL INTESTINES 19.4.1 Celiac Disease NAFLD patients have a higher incidence of celiac disease compared to the general population, despite celiac patients having a lower risk for metabolic syndrome before the start of a gluten-free diet. In turn, patients with celiac disease have a higher risk of developing NAFLD compared to the general population. In about 3.5% of patients with NAFLD, celiac disease represents the only extrahepatic manifestation of the disease. The incidence is highest in the frst year after diagnosis and was found to be persistently higher than in the general population over the ensuing 15 years (25). The pathophysiologic mechanism of this is unclear but may be related to cellular stress and apoptosis caused by celiac disease (26). Intestinal permeability is increased in both celiac disease and NAFLD, perhaps mediated by intestinal dysbiosis which has been shown to serve as a trigger for the development of NASH in patients with hepatic steatosis (27). 19.4.1.1 Small Intestinal Bacterial Overgrowth As the gut microbiome is altered in NAFLD (as previously discussed in Chapter 5), small intestinal bacterial overgrowth (SIBO) may also contribute to the pathogenesis of NAFLD. SIBO has been shown to be up to 65% more prevalent in the NAFLD than the non-NAFLD population (28). In one study evaluating liver biopsies of patients with NAFLD and SIBO, histological characteristics that were associated with SIBO included higher steatosis and fbrosis grade, lobular and portal infammation, and ballooning grade (29). In comparison with those without SIBO,

19 EXTRAHEPATIC GASTROINTESTINAL MANIFESTATIONS OF NONALCOHOLIC FATTY LIVER DISEASE

Figure 19.1

Possible mechanism explaining the increased prevalence of HP in patients with NAFLD

HP is purported to cause chronic low-grade systemic infammation, resulting in increased levels of infammatory cytokines such as IL-6 and TNF-α, which may affect insulin action and its level and thus promote insulin resistance. Additionally HP infection may also stimulate the release of leptin and adiponectin from adipose tissue, activating AMPK and upregulating SREBP1c and PPARα (22).

patients with SIBO had signifcantly higher endotoxin levels and higher CD14 mRNA, nuclear factor kappa beta mRNA, and toll-like receptor 4 (TLR4) protein expression. Patients with NASH had signifcantly higher endotoxin levels and higher intensity of TLR4 protein expression in comparison with patients without NASH (30). TLR expression in innate immune cells, such as dendritic cells and natural killer T cells, can trigger a pro-infammatory cascade leading to hepatocyte damage and the development of NASH. NASH subjects have elevated plasma levels of lipopolysaccharide associated with a rise in tumor necrosis factor (TNF)-α gene expression in the hepatic tissue. Additionally, SIBO is associated with increased gut permeability characterized by disruption of the intercellular tight junctions leading to bacterial translocation (31). Further studies are needed to determine whether therapy of SIBO reduces the risk of NAFLD, fbrosis and cirrhosis. 19.4.2 Infammatory Bowel Disease Various hepatic manifestations of IBD are well-described and beyond the scope of this chapter. However, in IBD patients with abnormal liver enzymes, NAFLD remains

the most common cause, affecting more than 30% of IBD patients (up to 55% of patients with UC and up to 40% of patients with Crohn’s disease) (32). NAFLD is more commonly seen in Crohn’s patients with a longer duration of IBD, older age, higher BMI and diabetes (33). A recent systematic review found prevalence rates up to 39.5% and from 1.5 to 55% in CD and UC patients, respectively (34). IBD behavior, extension, activity and drug response do not appear to be affected by the presence of NAFLD (35). IBD patients develop NAFLD with fewer metabolic risk factors than non-IBD NAFLD patients. The IBD-related intestinal infammatory state could be invoked to explain the higher prevalence of NAFLD in this population. In IBD, infammation causes breakdown of the gut barrier, leading to the translocation of bacteria and the release of cytokines (such as TNF alpha). These mediators may contribute to the pathophysiology behind NAFLD in such patients. Furthermore, alteration of the gut microbiome, welldescribed in IBD, may play a role in NAFLD progression in these patients (36,37). In one study, among 223 NAFLD patients, 78 patients with IBD were younger, were less likely to have altered liver enzymes, had lower mean body 177

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weight, smaller waist circumference and lower BMI and a lower prevalence of metabolic syndrome. Within the IBD population, patients with severe IBD and also those with a lower BMI had a higher prevalence of S3 on transient elastography (see discussion of lean NAFLD in Chapter 35). Independent risk factors for S3 in IBD patients were more than one IBD relapse per year during disease history, surgery for IBD and more extensive intestinal involvement. Anti-TNF therapy was the only independent factor found to be protective against abnormal liver enzymes. In another study, higher CDAI scores were seen among patients with NAFLD. Therefore, NAFLD in IBD patients appears to be different from that in non-IBD patients (38,39). IBD patients appear to develop NAFLD as a result of an increased infammatory load and not because of metabolic risk factors. 19.5 NAFLD AND THE COLON 19.5.1 Irritable Bowel Syndrome There is increasing interest in the possibility of an association between NAFLD and irritable bowel syndrome (IBS) due to similar etiologic factors for both diseases. The prevalence in the literature of both NAFLD and IBS occurring concomitantly is variable and ranges from 12 to 74%. One similar risk factor for both NAFLD and IBS is obesity. There is evidence that IBS is more prevalent in patients who are obese. It has been noted that increased

visceral adiposity enhances perception of luminal stimuli, dysmotility and abdominal pain. Higher body mass index has been associated with accelerated colonic and rectosigmoid transit and increased stool frequency. Additionally, dysregulation of the microbiome has been shown to be a component for the development of both NAFLD, as previously discussed, and IBS (40). As in NAFLD, altered intestinal motility and sensitivity, reduced microbial diversity, and the presence of a pro-infammatory state are also implicated in IBS (Figure 19.2). As depression and anxiety are both more prevalent in IBS and bowel symptoms signifcantly impact quality of life, a multidisciplinary approach may be needed for NAFLD patients who have coexistent IBS. 19.5.2 Diverticular Disease Both NAFLD and diverticular disease are associated with metabolic syndrome. In one study, an increased severity of diverticulitis was associated with the presence of hepatic steatosis. In a retrospective study investigating accompanying diseases in patients with NAFLD, Kempin´ski et al. found that colonic diverticulosis is the second most frequent concomitant gastrointestinal disease, second only to GERD. Colonic diverticulosis was signifcantly more prevalent in the study group than in the controls (23.7 vs. 15.8%). In addition to shared factors such as obesity, diets low in fber and high in fats and red meat, and altered gut

Figure 19.2 Several etiologic factors overlap between IBS and NAFLD: obesity, alterations in the gut microbiome, dietary factors and immune-mediated causes (41) 178

19 EXTRAHEPATIC GASTROINTESTINAL MANIFESTATIONS OF NONALCOHOLIC FATTY LIVER DISEASE

microbiome, arterial hypertension has importantly also been implicated in the development of both NAFLD and diverticulosis. Arterial hypertension can over time lead to structural changes in the bowel wall by damaging blood vessels and further reducing blood supply in vulnerable anatomical areas (42). 19.5.3 Clostridioides diffcile NAFLD has been shown to be associated not only with Clostridioides diffcile infection (CDI) but also recurrent CDI. Hospitalized CDI patients with NAFLD in one study had more intestinal complications with either perforation or obstruction compared to CDI patients with liver disease of another etiology such as viral hepatitis or alcoholic liver disease. On the other hand, NAFLD was found to be an independent predictor of CDI in another study (43). Gut dysbiosis may contribute to the pathogenesis of CDI as in NAFLD. Bacteroides and Bifdobacterium play an important role in preventing colonization by C. diffcile, and patients with NAFLD were shown to have a relative decrease in the proportion of Bacteroides to Firmicutes. The chronic low-level infammation associated with NAFLD and the resultant impaired immune response might predispose patients to CDI as well as other infections. 19.5.4 Colonic Adenomas and Colorectal Cancer NAFLD is associated with an increased risk of developing colorectal adenomatous polyps. This is true even after adjusting for diabetes, obesity and hyperlipidemia. Furthermore, adenomatous polyps are more common in those NAFLD patients with advanced fbrosis compared to no fbrosis and in those with NASH compared to simple steatosis (44). In a retrospective cohort study, Hwang et al. evaluated 2,917 patients who had undergone colonoscopy and found an ultrasound-diagnosed NAFLD prevalence of 41.5% in the patients with adenomas compared to 30.2% among patients without adenomas, and this was independent of the presence of metabolic syndrome (45). A population-based study in Korea and prospective study in China have shown an increased prevalence of not only colonic adenomas but also of advanced neoplasia such as colonic cancer, high-grade dysplasia and villous histology in patients with NAFLD (46,47). In one large cohort study, sigmoid colon adenocarcinoma and highly differentiated colorectal adenocarcinoma were more common among NAFLD patients (48). Lifestyle factors, such as a high-fat/low-fber diet, increased consumption of red processed meat and a sedentary lifestyle, can cause insulin resistance and alter the gut microbiome. These, in turn, can cause mitochondrial dysfunction, increased oxidative stress, intestinal barrier destruction and increased production and absorption of gut-derived toxic and pro-infammatory metabolites, such as advanced glycation end products (AGEs), IL-6, IL-8 and plasminogen activator inhibitor-1 (PAI-1). These proinfammatory metabolites have been implicated in the pathogenesis of NAFLD and also disrupt intestinal cell repair and apoptosis, thus potentiating tumorigenesis (49). Furthermore, NAFLD patients have disordered adipocytokine metabolism. Typically, adiponectin has anticarcinogenic effects as it suppresses cell growth of carcinoma through the AMPc-activated protein kinase. Decreased adiponectin and increased leptin serum levels have been demonstrated in NAFLD patients. These hormones, secreted by adipocytes, have been proposed to play an important role in the development of colorectal cancer (50).

19.6 NAFLD AND THE PANCREATICOBILIARY TRACT 19.6.1 Biliary Disease and Cancer NAFLD and gallbladder disease share similar risk factors, such as obesity, insulin resistance, dyslipidemia, and high dietary cholesterol intake. In particular, female sex, increased age, higher BMI, and the presence of NASH and fbrosis are associated with the development of cholelithiasis in NAFLD patients. In one meta-analysis, the pooled prevalence of gallbladder disease in NAFLD patients was 17%, which is higher than in the general population (51). In other studies, the prevalence of cholelithiasis in NAFLD patients was found to be as high as 47% (52). It is wellknown that in obesity, diabetes and hypertriglyceridemia, secretion of biliary cholesterol as well as gallbladder motility are disrupted and that biliary composition and gallbladder emptying are affected. At the cellular level, NAFLD patients were found to have a decreased level of farnesoid X receptor and its mRNA in liver tissue. This nuclear receptor plays an important role in regulating the transcription of ATP-binding cassette transporters on the hepatocyte canalicular membrane, resulting in decreased activity of bile salt export pumps. Consequently, there is a decrease in biliary concentration of bile acids and phospholipids, leading to reduced solubility of cholesterol and thus increased risk of gallstone formation. Biliary tract cancers, including cholangiocarcinoma (CCA) and gallbladder cancer, are the second most common type of hepatobiliary cancer worldwide. NAFLD is associated with an increased risk of CCA, particularly intrahepatic CCA, and the risk is further increased in NASH patients (53,54). This is primarily thought to occur via the induction of hepatic infammation. As with colorectal cancer, it is mediated through NAFLDassociated gut dysbiosis, which can promote carcinogenesis via the gut-liver axis. The hypothesized mechanism consists of increased intestinal permeability and hepatic production and systemic release of numerous proinfammatory cytokines that promote carcinogenesis through proliferation, antiapoptosis and angiogenesis. This effect appears to be independent of other factors such as obesity (55). 19.6.2 Pancreatitis NAFLD has been associated with acute pancreatitis (AP), independent of obesity. In hospitalized patients, the presence of NAFLD was shown to increase the risk of death, more severe AP, necrotizing pancreatitis, SIRS and length of hospital stay. Furthermore, in one study, NAFLD was found to be a risk factor for postendoscopic retrograde cholangiopancreatography pancreatitis. Such patients may beneft from prophylactic pancreatic stenting and/or rectal NSAID therapy (56). 19.6.3 Pancreatic Cancer NAFLD may be an important risk factor for pancreatic cancer, presumably related to high BMI and visceral adiposity. In one study, NAFLD was also found to be an independent risk factor for pancreatic cancer and was associated with decreased survival following diagnosis. The role of NAFLD in pancreatic cancer may be driven by dysregulated cytokine activity, promoting the progression of pancreatic cancer. Insulin resistance results in the release of pro-infammatory cytokines from excess. Cytokines, such as adiponectin, IGF-1, TNF-α, IL-6 and 179

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VEGF, contribute to the development of cancer through angiogenesis, cell migration and mitogenesis (57). 19.7 SUMMARY Nonalcoholic fatty liver disease (NAFLD) is associated with a variety of extrahepatic gastrointestinal manifestations. The prevalence of esophageal, gastric, small bowel, colonic and pancreaticobiliary diseases in this patient population has important implications in the management of NAFLD as a multisystem disease. Alterations in the gut microbiome, insulin resistance and the release of proinfammatory cytokines may account for these associated conditions. Further studies are needed to determine the optimal methods by which to manage NAFLD patients as they pertain to extrahepatic manifestations of the disease, including the potential impact on surveillance for malignancy among patients with NAFLD. REFERENCES 1. Miele L, Cammarota G, Vero V, Racco S, Cefalo C, Marrone G, Pompili M, Rapaccini G, Bianco A, Landolf R, Gasbarrini A, Grieco A. Non-alcoholic fatty liver disease is associated with high prevalence of gastro-oesophageal refux symptoms. Dig Liver Dis. 2012;44(12):1032–1036. doi:10.1016/j.dld.2012.08.005. Epub 2012 Sep 7. PMID: 22963909 2. Hung WC, Wu JS, Yang YC, Sun ZJ, Lu FH, Chang CJ. Nonalcoholic fatty liver disease vs. obesity on the risk of erosive oesophagitis. Eur J Clin Invest. 2014;44(12):1143–1149. doi:10.1111/eci.12348. Epub 2014 Nov 3. PMID: 25293867 3. Yang HJ, Chang Y, Park SK, Jung YS, Park JH, Park DI, Cho YK, Ryu S, Sohn CI. Nonalcoholic fatty liver disease is associated with increased risk of refux esophagitis. Dig Dis Sci. 2017;62(12):3605–3613. doi:10.1007/ s10620-017-4805-6. Epub 2017 Oct 23. PMID: 29063416 4. Mikolasevic I, Poropat G, Filipec Kanizaj T, Skenderevic N, Zelic M, Matasin M, Vranic L, Kresovic A, Hauser G. Association between gastroesophageal refux disease and elastographic parameters of liver steatosis and fbrosis: controlled attenuation parameter and liver stiffness measurements. Can J Gastroenterol Hepatol. 2021;2021:6670065. doi:10.1155/2021/6670065. PMID: 33688490; PMCID: PMC7925017 5. Taketani H, Sumida Y, Tanaka S, Imajo K, Yoneda M, Hyogo H, Ono M, Fujii H, Eguchi Y, Kanemasa K, Chayama K, Itoh Y, Yoshikawa T, Saibara T, Fujimoto K, Nakajima A, Japan Study Group of NAFLD. The association of insomnia with gastroesophageal refux symptoms in biopsy-proven nonalcoholic fatty liver disease. J Gastroenterol. 2014;49(7):1163–1174. doi:10.1007/ s00535-013-0871-5. Epub 2013 Aug 22. PMID: 23975270 6. Spiegel K, Leproult R, L’Hermite-Balériaux M, Copinschi G, Penev PD, Van Cauter E. Leptin levels are dependent on sleep duration: relationships with sympathovagal balance, carbohydrate regulation, cortisol, and thyrotropin. J Clin Endocrinol Metab. 2004;89: 5762–5771. doi:10.1210/jc.2004-1003 7. Akiyama T, Yoneda M, Inamori M, Iida H, Endo H, Hosono K, Yoneda K, Fujita K, Koide T, Tokoro C, Takahashi H, Goto A, Abe Y, Kirikoshi H, Kobayashi N, Kubota K, Saito S, Nakajima A. Visceral obesity and the risk of Barrett’s esophagus in Japanese patients with non-alcoholic fatty liver disease. BMC Gastroenterol. 2009;9:56. doi:10.1186/1471-230X-9-56. PMID: 19622165; PMCID: PMC2718904 180

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20 EXTRAHEPATIC AND HEPATIC CANCERS

20 Extrahepatic and Hepatic Cancers Maryam Ibrahim and Tracey G. Simon

CONTENTS 20.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 20.2 Carcinogenesis in NAFLD: Potential Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 20.2.1 Hepatic Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 20.2.2 Extrahepatic Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 20.3 NAFLD and GI Malignancies: Clinical and Epidemiological Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 20.3.1 Hepatocellular Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 20.3.2 HCC Surveillance/Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 20.3.3 Challenges and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 20.3.4 Intra- and Extrahepatic Cholangiocarcinoma (CCA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 20.3.5 Colorectal Cancer (CRC) and Adenomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.3.6 CRC Screening in Patients with NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.3.7 Liver Metastasis from CRC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.3.8 Other GI Malignancies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.3.8.1 Esophageal, Gastric and Pancreatic CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.4 NAFLD and Non-GI Malignancies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.4.1 Renal Cell Carcinoma (RCC). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 20.4.2 Prostate CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 20.4.3 Breast CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 20.4.4 Uterine CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 20.4.5 Lung CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 20.4.6 Hematologic CA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 20.4.7 Is It Time to Change Our Extrahepatic Cancer Screening Strategies for Patients with NAFLD? . . . . . . . . . 190 20.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Key Points ■

NAFLD is a multisystem disease associated with extrahepatic complications and cancers.

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Carcinogenesis is mediated through changes in lipid metabolism, growth and proliferation pathways, oxidative stress, immune signaling, genetics and microbiome.

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Robust evidence is available for the increased incidence of HCC (hepatocellular carcinoma) in NAFLD.

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NAFLD has been linked to increased cholangiocarcinoma risk, with a stronger association for intrahepatic cholangiocarcinoma.

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There appears to be a higher prevalence of colorectal lesions in NAFLD.

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More evidence is still needed for associations between NAFLD and esophageal, gastric, pancreatic, renal, prostate, breast, uterine, lung and hematologic cancers.

20.1 INTRODUCTION NAFLD is now viewed as a multisystem disease that is associated with extrahepatic complications [1]. Increasing evidence now links NAFLD with various diseases, including diabetes, kidney disease, cardiovascular disease and cancer. Recently, there has been increased attention to the possible association between NAFLD and cancer development. Malignancies, whether within or outside the GI tract, represent signifcant contributors to death in patients with NAFLD [1]. While the precise mechanisms linking NAFLD to carcinogenesis are largely undefned, NAFLD is viewed as the hepatic manifestation of metabolic syndrome, and a body of evidence indicates an increased DOI: 10.1201/9781003386698-24

risk of cancer—particularly gastrointestinal and hepatic cancers—in patients with metabolic syndrome [2]. In this setting, NAFLD can either share common risk factors (such as obesity or type 2 diabetes), or it may actively mediate various pathogenic mechanisms, leading to cancer [2]. In this chapter, we review the current evidence and potential mechanisms underpinning observed associations between NAFLD and both hepatic and extrahepatic cancers (with a focus on cholangiocarcinoma, colorectal, esophageal, gastric, pancreatic, genitourinary, breast, uterine, lung and hematologic cancers). 20.2 CARCINOGENESIS IN NAFLD: Potential Mechanisms 20.2.1 Hepatic Cancers It has been hypothesized that the pathogenesis of cancer in patients with NAFLD is related to a complex range of environmental factors (lifestyle, diet, microbiome) acting upon a susceptible genetic or epigenetic background in order to modify responses to caloric excess [3]. Obesity, diabetes and the metabolic syndrome are essential elements of the association between NAFLD and HCC [4,5]. Dyslipidemia and hypertension, two additional components of metabolic syndrome, have also been shown to contribute to HCC risk in a recent analysis of US patients from the Veterans Healthcare Administration [4]. As such, NASH-HCC pathogenesis is infuenced by derangements that occur in the setting of obesity and the metabolic syndrome, which include oxidative stress and aberrant lipid metabolism [4]. In particular, the excess accumulation of intrahepatic lipids promotes mitochondrial dysfunction and oxidative stress, which in turn ignites a cascade of carcinogenic and pro-infammatory processes including changes in 183

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lipid metabolism, activation of growth and proliferation pathways, as well as changes in the immune response [4] (see Figure 20.1). While the precise mechanism linking obesity and the metabolic syndrome to the pathogenesis of NAFLDHCC is still undefned, several have been proposed. One proposed mechanism is through changes in lipid metabolism. In order for hepatocytes to escape lipotoxicity, they downregulate carnitine palmitoyltransferase 2 (CPT2), which drives malignant transformation through the accumulation of acylcarnitine [6]. Moreover, sterol regulatory element-binding proteins transcription factors (SREBPs), which are known to interact with p53, are modifed in the setting of oxidative stress, and this may contribute to hepatocarcinogenesis [7] (see Figure 20.1). NAFLD-related HCC may also arise through changes in growth and proliferation pathways, which are directly affected by the metabolic syndrome. Fatty acid accumulation induces junctional protein associated with coronary artery disease (JCAD), a protein which promotes the progression of NASH to HCC by inhibiting LATS2 kinase activity [8]. The Hedgehog (Hh) pathway is consistently activated in NASH, with a level of activity that correlates with liver disease severity, and increased Hh pathway activity has also been observed in hepatic carcinogenesis

Figure 20.1

[9]. Insulin resistance and insulin-like growth factor (IGF) signaling have also been implicated in NASH-HCC. Insulin resistance and subsequent hyperinsulinemia increase the activity of IGF-1, which stimulates hepatocyte growth via Akt and mTOR, potentially driving liver carcinogenesis [10]. IGF-1 demonstrates antiapoptotic effects in NASH animal models, but the paradoxical upregulation of IGF-1R in HCC tissue suggests that the antiapoptotic effect might be driving abnormal growth [11]. Oxidative stress induces caspase-2, which drives apoptosis and the subsequent compensatory proliferation of hepatocytes [12,13]. Finally, it has been proposed that oxidative stress could promote hepatocarcinogenesis by impacting immune signaling pathways [4]. Specifcally, CD4+T cells which play a pivotal role in antitumor surveillance are selectively lost due to susceptibility to oxidative stress [14]. Obesity-related chronic infammation activates Kupffer cells through TREM1 receptor, which drives the secretion of infammatory cytokines such as IL-6 and TNF-a. This causes alterations in NF-kB signaling, hepatocyte proliferation and subsequent HCC development [15,16] (see Figure 20.1). Other mechanisms implicated in NASH-HCC development include genetic variants and gut microbiota [4]. Genetic variants associated with HCC include patatin-like

Potential mechanisms for carcinogenesis in NAFLD

PNPLA3: Patatin-like phospholipase domain-containing protein 3; TERT: Telomerase reverse transcriptase; Apo B: Apolipoprotein B; FXR: Farnesoid X receptor; TMAO: Trimethylamine N-oxide; ROS: Reactive oxygen species; CPT2: Carnitine palmitoyltransferase 2; SREBP: Sterol regulatory element-binding proteins; JCAD: Junctional protein associated with coronary artery disease; Hh: Hedgehog; IGF-1: Insulin-like growth factor-1; Akt: Protein kinase B; mTOR: Mammalian target of rapamycin; IL-6: Interleukin-6; NF-kB: Nuclear factor kappa light chain enhancer of activated B cells.

184

20 EXTRAHEPATIC AND HEPATIC CANCERS

phospholipase domain-containing protein 3 (PNPLA3) polymorphisms, telomerase reverse transcriptase (TERT) promoter mutations and apolipoprotein B mutations, among others [17–19]. Moreover, increasing evidence suggests the role of gut dysbiosis in cirrhosis and subsequent HCC development. In a study of patients with NASH cirrhosis, patients with HCC had increased levels of Bacteroides and Ruminococcaceae, whereas Akkermansia and Bifdobacterium were reduced in comparison to NASHcirrhosis without HCC [20]. NASH-HCC has also been linked to dysbiosis through bile acid signaling to liver cells, which triggers upregulation in growth pathways such as mTOR [21,22]. The gut microbiome regulates bile acids and consequently the farnesoid X receptor (FXR), a bile acid sensor [23]. FXR helps to prevent hepatocarcinogenesis by protecting against bile acid-related liver injury and modulating fbrosis in mouse models [23]. Further studies are needed to investigate the role of FXR signaling in metabolic syndrome, NASH, and NASH to HCC transition [24]. Other metabolites through which the gut microbiome could contribute to HCC development include trimethylamine (TMA) and its metabolite trimethylamine N-oxide (TMAO) as well as ethanol, which are generated by gut bacteria [22]. High TMAO levels are associated with insulin resistance, suggesting that it could contribute to NAFLD progression and possibly NASH-HCC [22]. Ethanol, a known hepatotoxin and carcinogen, is produced by gut microbiota and is increased in patients with NASH [22]. Whether this contributes to NAFLD progression and HCC development is currently unknown (see Figure 20.1). 20.2.2 Extrahepatic Cancers Several mechanisms have been suggested to link NAFLD to extrahepatic cancers, mainly through obesity-related chronic infammation, dysbiosis, insulin resistance and changes in adiponectin and leptin levels [4,25]. However, the exact mechanism by which NAFLD might promote extrahepatic tumorigenesis remains unclear. It was suggested that visceral adipose tissue accumulation in NAFLD may affect other organs by releasing cytokines such as growth factors, adipocytokines and proinfammatory factors [26]. Emerging data support the fact that local ectopic fat may affect the paracrine pathway to induce cancer development in the pancreas and breast [27,28]. However, these fndings remain preliminary, and future research is needed to precisely defne the mechanisms and pathways that might link NAFLD to the development of extrahepatic cancers. 20.3 NAFLD AND GI MALIGNANCIES: Clinical and Epidemiological Data 20.3.1 Hepatocellular Carcinoma HCC is one of very few cancers with growing incidence and mortality in the US and worldwide [29], and it now represents the fourth leading cause of cancer death in the world [30,31]. In parallel with the growing epidemics of obesity and type 2 diabetes, NAFLD is a growing cause of HCC in the US [4]. Studies from South Korea, Europe and southeast Asia have shown a similar rapid increase in NAFLD-HCC over the last two decades [23]. In the US, it is now the fastest growing cause of HCC among listed liver transplant candidates as well as transplant recipients [32]. In cohorts of patients with NASH in Europe and the US, the annual incidence of HCC ranges from 0.7 to 2.6% [33,34]. The incidence of HCC in patients with NASH is higher in men, older patients, Hispanics and patients with

higher alcohol intake and diabetes [5,33,35]. A prospective cohort of biopsy-proven NASH cirrhosis from Japan had an 11.3% 5-year incidence of HCC [36]. This cohort was older and had a higher prevalence of diabetes, which could account for the higher observed HCC incidence [23]. In a population-based cohort study conducted by Simon et al. including 8,892 adults with histologically defned NAFLD in Sweden, NAFLD patients had signifcantly increased overall cancer incidence (10.9 vs. 13.8/1,000 person-years [PYs]; aHR, 1.27 [95%CI, 1.18–1.36]), driven primarily by HCC (aHR, 17.08 [95%CI, 11.56–25.25]) [37]. The mean age of NAFLD-related HCC is 73 years, which is higher than the mean age of patients with viral hepatitis-related HCC [38]. NASH-HCC is diagnosed at a later stage, and the mortality appears to be higher than HCC from other liver conditions [38,39]. The lack of public awareness surrounding NAFLD and its progression, as well as the lack of HCC screening guidelines in high-risk patients with NAFLD without cirrhosis contributes to the late diagnosis and treatment [23]. It is estimated that 3–5% of the current adult US population have NASH, with a projected increase in the prevalence rate by 2030 [40]. It has also been reported that HCC may arise in patients with NASH even in the absence of cirrhosis; thus the rising NASH prevalence is quite alarming [4]. In two large studies in the Veterans Healthcare Administration, 20 to 36% of NASH-HCC cases did not have underlying cirrhosis, and NAFLD was the leading etiology of HCC in patients without cirrhosis [35,41]. An Italian multicenter cohort of patients with NAFLD-HCC found that 50% of the patients were without cirrhosis but rather had advanced fbrosis [39]. This suggests that the fbrosis stage might be relevant in future efforts to risk-stratify NAFLD patients according to their future HCC risk [23]. Similarly, in a Japanese multicenter cohort, 72% of the patients with biopsy-proven NAFLD-HCC had advanced fbrosis (F3 and F4) [42], further underscoring that HCC may develop in NASH with advanced fbrosis [23]. The proportions of patients with HCC attributable to NAFLD varies widely across different counties/regions, with percentages ranging between 1 and 38% [23], due in large part to discrepancies in defnitions of NAFLD and its ascertainment [23]. Several US studies have used either primary diagnoses reported in national transplantation databases or ICD-9 codes from large national registries [23]. Many studies have expanded their study defnitions to include patients with cryptogenic cirrhosis and components of the metabolic syndrome, as NAFLD is often underreported [43,44]. Some studies also include patients with cryptogenic cirrhosis even without the metabolic syndrome [45]. However, a notable prior study by Thuluvath et al., looking at the United Network for Organ Sharing database, found that patients with cryptogenic cirrhosis have a different risk factor profle than patients with NASH cirrhosis and concluded that NASH cirrhosis and cryptogenic cirrhosis are not equivalent [46]. Further adjustment in the defnitions of suspected cryptogenic cirrhosis and NASH cirrhosis would be important for a more precise estimate of the proportion of patients with NAFLD-HCC without cirrhosis, as well as the risk of HCC in patients with cirrhosis of either etiology [23]. Several studies around the world have specifcally investigated the relationship between diabetes and HCC risk among patients with NAFLD. In a Mayo Clinic study, the presence of diabetes was associated with a 4-fold increase 185

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in the risk of HCC in patients with NASH cirrhosis. This was validated using liver transplant registrants identifed within the United Network for Organ Sharing, whereby diabetes was an independent risk factor for HCC development in patients with NASH cirrhosis [5]. Diabetes was also the strongest independent risk factor for HCC development in patients with NAFLD in a large European population-based study [47]. Moreover, Kanwal et al. found that among patients with NAFLD, the presence of additional, comorbid metabolic traits led to a stepwise increase in HCC risk and that diabetes was associated with the highest risk of development of HCC [48]. Collectively, these fndings underscored the importance of regular screening for diabetes and rigorous attention to blood sugar control, among patients with NAFLD [23]. 20.3.2 HCC Surveillance/Screening NASH-HCC has a unique natural history and pathogenesis that makes HCC surveillance in patients with NASH quite challenging [4]. There is no current high-level, NAFLD-specifc evidence to support or refute the clinical impact and cost-effectiveness of established HCC surveillance protocols in the setting of NAFLD or NASH cirrhosis [23]. Accordingly, it is recommended that all patients with NAFLD cirrhosis—as in all etiologies of cirrhosis—undergo regular semiannual HCC screening [49]. However, there is increasing attention paid to the need for more personalized HCC surveillance protocols. Importantly, as NAFLD patients without cirrhosis can develop HCC, HCC surveillance might be considered in selected, high-risk patients with advanced fbrosis on a case-by-case basis [23]. The American Gastroenterology Association (AGA) Clinical Practice Update recommends considering HCC screening in patients with noninvasive markers that indicate the presence of advanced fbrosis (F3) or cirrhosis [50]. The AGA recommends combining 2 or more noninvasive fbrosis tests of separate categories (imaging based and blood based) in order to minimize the likelihood of misclassifcation [23]. If the test results are concordant for either advanced fbrosis or cirrhosis, the AGA recommends consideration of HCC surveillance [23]. The European Association for the Study of the Liver (EASL) guidelines recommend individual risk assessment for HCC surveillance in patients with liver disease of any etiology with advanced fbrosis (F3) [51]. No specifc recommendations for HCC surveillance in NAFLD patients without cirrhosis are provided by the Asian Pacifc Association for the Study of Liver disease (APASL) clinical practice guidelines [52,53]. Patients without advanced fbrosis have too low an HCC risk, and as such routine screening is not currently recommended [35]. The recommended modality for HCC surveillance in the AASLD, APASL and EASL practice guidelines is ultrasonography with or without concomitant alpha-fetoprotein level [51,52,54]. In a study of 941 patients with cirrhosis undergoing ultrasonography, the quality was inadequate to exclude liver lesions in 20% of the scans [55]. Elevated BMI and NASH cirrhosis were independent factors associated with inadequate quality of scans [55]. Thus it is imperative for future clinical and implementation research to focus on improving both the methods of assessment and the reporting of the quality of HCC surveillance ultrasonography in order to improve early detection of NAFLDHCC tumors. 186

20.3.3 Challenges and Future Prospects There continues to be a debate over the cost-effectiveness and potential harm from overinvestigating false positive tests for HCC screening [24]. The debate is especially pertinent for NAFLD patients, as they are more likely to have unrecognized liver disease and are at high risk of having low-quality imaging studies for HCC screening. Even in known NAFLD-associated cirrhosis, surveillance is less likely to detect early NAFLD-associated HCC than HCC arising from other etiologies [24]. This is also relevant when considering initiation of HCC surveillance in patients with advanced NAFLD, who do not have cirrhosis. This represents an enormous and growing population, which is exposed to a relatively small—albeit devastating—risk, and in that setting it has thus far been felt that the cost of ultrasound surveillance is prohibitive [24]. Currently, the hope is that further research will identify combinations of factors that can better risk-stratify patients with NAFLD, in order to create more targeted, cost-effective surveillance strategies [24]. To that end, the combination GALAD serum score has shown promising diagnostic performance, and its prospective evaluation as an HCC surveillance biomarker is eagerly awaited [24]. Other candidates awaiting validation include the serum glycoproteins osteopontin and dickkopf (24), panels of nucleic acids and extracellular vesicles (EVs), as well as circulating tumor DNA (ctDNA) [24] or proteomic signatures in serum EVs [24]. Another challenge arises in the management of patients with NAFLD-associated HCC. The Barcelona Clinic for Liver Cancer (BCLC) is the algorithm employed in the AASLD and EASL guidelines, which guide staging and treatment selection [24]. Unfortunately, it is rare to detect NAFLD-HCC at the BCLC-0 or BCLA-A stage [24]. Even when the tumor is small, advanced age and metabolic syndrome-associated comorbidity make curative procedures more challenging. Rather, the vast majority of NAFLDHCC are classifed as BCLC-C or BCLC-D, owing largely to the performance status test (PST) [24]. PST has a major effect on survival; however, it is a subjective measure that can be more diffcult to assess in patients with metabolic syndrome [24]. Thus improved strategies for early detection and preventive strategies remain an urgent necessity to avoid the alarming projected increases in NAFLD-HCC incidence and mortality. The ongoing challenge for all patients with HCC, including those with NAFLD-HCC, will be to identify robust biomarkers that will improve risk stratifcation and guide both treatment selection and monitoring 20.3.4 Intra- and Extrahepatic Cholangiocarcinoma (CCA) NAFLD has been linked to increased CCA risk, with some studies indicating a stronger association with intrahepatic CCA, suggesting underlying common mechanisms to HCC [56]. In a systematic review and meta-analysis by Liu et al., which included studies from USA, Europe and Asia, NAFLD was associated with a signifcantly higher risk of developing both intrahepatic and extrahepatic CCA [1]. Another meta-analysis by Wongjarupong et al. included seven case-control studies with a total of 9,102 CCA patients and observed similarly increased intrahepatic and extrahepatic CCA risk in patients with NAFLD [56]. Despite the preceding meta-analyses, the association between NAFLD and extrahepatic CCA is still

20 EXTRAHEPATIC AND HEPATIC CANCERS

controversial. Recently, Corrao et al. performed a meta-analysis as well as trial sequential analyses to study the association between NAFLD and CCA [57]. They found that NAFLD determines an increased risk of total as well as intrahepatic CCA incidence, but they observed no association for extrahepatic CCA [57]. The subsequent trial sequential analyses proved that the associations were conclusive, and the authors thereby concluded that there is no association between NAFLD and extrahepatic CCA [57]. As such, further studies are still needed to study the existence or magnitude of this association. 20.3.5 Colorectal Cancer (CRC) and Adenomas To date, the majority of prior studies has found a higher prevalence of colorectal lesions in patients with NAFLD [2]. Hwang et al. provided the frst evidence for the association between colorectal adenomatous polyps and NAFLD [58]. In their study of 2,917 patients, NAFLD was more prevalent in the adenomatous polyp group as compared to controls, and it was associated with 3-fold increased odds of colorectal adenomas [58]. These fndings were confrmed in a large Korean retrospective cohort study, whereby NAFLD patients had a 3-fold increase in the risk of CRC and a 2-fold increase in the occurrence of polyps [59]. In a cross-sectional study by Wong et al., the presence of NASH was associated with the highest CRC risk, among NAFLD patients [60]. Although they further reported a high prevalence of right-sided CRC [60], it remains unclear whether NAFLD or its histological features are linked to specifc CRC subtypes, and larger, prospective studies are still needed to clarify this question. Longitudinal studies have largely replicated these fndings [2]. For example, the incidence of de novo adenoma was increased by 45% in patients with NAFLD in a prospective study where patients had paired colonoscopies, with negative index colonoscopies [61]. In a Danish cohort study of hospitalized patients, an increased risk of CRC was seen with fatty liver as compared to the general population [62]. A recent meta-analysis of 10 studies showed that NAFLD patients have a signifcantly higher risk of developing colorectal adenomas and CRC [1]. In another meta-analysis, the presence and severity of NAFLD were again associated with an increased risk of incident CRC or adenomas [63]. Finally, in asymptomatic adults undergoing a screening colonoscopy, a meta-analysis by Mantovani et al. found similar positive associations for colorectal adenoma and cancer [64]. In contrast, only two studies failed to demonstrate a statistically signifcant increase in the incidence of colorectal adenomas in patients with NAFLD [2]. 20.3.6 CRC Screening in Patients with NAFLD Given the observed associations between NAFLD, NASH and the formation of adenomatous colon polyps, including right-sided polyps, polyps with high-grade dysplasia and CRC [2,60], some authors have suggested enhanced CRC screening and surveillance in this population [60]. However, to date the available body of evidence is still limited, and it is unclear whether patients with NAFLD would derive beneft from early or increased CRC screening and surveillance strategies. Thus, at present, it is important for clinicians to continue recommending that patients with NAFLD undergo appropriate CRC screening as per published guidelines [65].

20.3.7 Liver Metastasis from CRC The “seed-soil” hypothesis suggests that metastatic cancer cells will migrate to an area where the local microenvironment is favorable. The “soil” has unique biological characteristics and microenvironment with special molecular components and cells that promote metastasis formation [66]. Several studies have addressed the role of the liver with NAFLD as “soil” in the metastasis of CRC cells as “seeds” [66]. Yan et al. found that in patients with CRC, the presence of NAFLD was associated with an increased incidence of synchronous liver metastasis, detected on or prior to the primary CRC tumor [66]. These fndings are supported by preclinical data demonstrating that hepatic steatosis increases colon tumor metastasis to the liver [67,68]. However, other studies have found that liver metastases of CRC arise less frequently in the setting of NAFLD [69,70]; thus prospective research is still needed in populations with well-phenotyped NAFLD, which also carefully distinguishes between synchronous and metachronous CRC metastasis [66,69,70]. 20.3.8 Other GI Malignancies 20.3.8.1 Esophageal, Gastric and Pancreatic CA While central adiposity may represent an important, shared risk factor for NAFLD and for esophageal adenocarcinoma [71], a link between NAFLD and esophageal or gastric cancer is less clear. Although very few clinical studies have studied these links, it has been reported that NAFLD is associated with the risk of developing esophageal cancer [1], and a meta-analysis of 3 high-quality studies suggested that NAFLD patients have an elevated risk of developing gastric cancer [1]. However, most prior studies to date have been limited by small sample sizes with few cancer outcomes or by a limited ability to accurately phenotype NAFLD and its severity [71]. More recently, in a large retrospective analysis of patients with biopsy-confrmed NAFLD, Simon et al. found no signifcant association between NAFLD and incident gastric or esophageal cancer [37]. Pancreatic cancer has been shown to be highly associated with NAFLD [71]. A meta-analysis of 3 high-quality studies suggested that NAFLD patients have an elevated risk of developing pancreatic cancer [1,72,73], while a more recent retrospective study showed signifcant higher crude incidence but nonsignifcant adjusted risk [74]. Emerging epidemiologic and translational data support the role of local ectopic fat as a paracrine mechanism for the development of pancreatic cancer, where the local adipose tissue microenvironment impacts tumor progression. As such, it would be biologically plausible for NAFLD to contribute to the development of pancreatic cancer, and further research is needed to better characterize these potential associations [73] (see Table 20.1). 20.4 NAFLD AND NON-GI MALIGNANCIES 20.4.1 Renal Cell Carcinoma (RCC) To date, NAFLD has been linked to increased RCC incidence in 3 large retrospective cohort studies [62,72,74], but only one study showed a statistically signifcant risk [62,71]. Watanabe et al. found that in Japanese populations, NAFLD might be associated with more severe RCC and shorter overall survival [75]. In their large retrospective study, Simon et al. found that NAFLD patients had signifcantly higher rates of developing kidney/bladder cancer [37]. Further research in large populations is still needed to better clarify this potential relationship (see Table 20.1). 187

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Table 20.1: Summary of Studies That Investigated the Association between NAFLD and Extrahepatic Cancers Extrahepatic Cancer

Study

Type of Study

Colorectal

Hwang S. T. et al., (2009) [58]

Cross-sectional

South Korea

Colorectal

Stadlmayr A. et al., (2011) [79] Wong V. W. et al., (2011) [60]

Cross-sectional

Austria

Cross-sectional

China

Lee Y. I. et al. (2012) [59] Huang K. W. et al. (2013) [61]

Retrospective cohort Retrospective Cohort

South Korea Taiwan

Colorectal

Lin X. F. et al. (2014) [80]

Cross-sectional

China

Colorectal

Sun L. M. et al. (2015) [74] Bhatt B. D. et al.,(2015) [81]

Retrospective cohort Retrospective cohort

Taiwan

Lee T. et al. (2016) [82] Yang Y. J. et al. (2017) [83]

Cross-sectional

South Korea South Korea

Colorectal

Ahn J. S. et al., (2017) [84]

Cross-sectional

South Korea

Colorectal

Kim G. A. et al., (2017) [72] Kim M. C. et al. (2019) [85]

Retrospective cohort CrossSectional

South Korea South Korea

Colorectal

Cho Y. et al. (2019) [86]

Cross-sectional

South Korea

Colorectal

Allen A. M., et al. (2019) [73] Simon TG, et al. (2021) [37] Sorensen HT et al. (2003) [62] Uzel M et al. (2015) [87]

Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort

USA

Colorectal

Colorectal Colorectal

Colorectal

Colorectal Colorectal

Colorectal

Colorectal Esophageal Gastric

188

Retrospective cohort

Country

USA

Sweden Denmark Turkey

Findings NAFLD was associated with increased risk of colorectal adenomas (OR 1.28,95% CI: 1.03–1.6). Higher prevalence of NAFLD among adenoma group vs. nonadenoma group (41.5 vs. 30.2%). Independent association between NAFLD and increased risk of colorectal adenomas (aOR 1.47, 95% CI 1.08–2). Higher prevalence of colorectal adenomas (34.7 vs. 21.5%), as well as advanced neoplasms (18.6 vs. 5.5%) in NAFLD group as compared to healthy controls. Among the biopsy group, there was higher prevalence of adenomas (51 vs. 25.6%) and advanced neoplasms (34.7 vs. 14%) in patients with NASH as compared to those with simple steatosis. NASH was associated with adenomas (aOR 4.89, 95% CI 2.04–11.7) and advanced neoplasms (OR 5.34, 95% CI 1.92–14.84). NAFLD was associated with adenomatous polyps (aRR 1.94, 95% CI 1.11–3.4) and colorectal cancer (aRR 3.08, 95% CI 1.02–9.34). Higher prevalence of NAFLD in the adenoma group as compared to the nonadenoma group (55.6 vs. 38.8%). After adjusting, NAFLD was an independent risk factor for development of colorectal adenoma (OR 1.45; 95% CI: 1.07–1.98). Higher prevalence of colorectal cancer in NAFLD group (29.3 vs. 18%). NAFLD was independently associated with increased colorectal cancer risk (aOR 1.87, 95% CI 1.36–2.57). Higher incidence of colorectal cancer in NAFLD with cirrhosis group (aHR 2.58, 95% CI 1.59–4.18). Higher prevalence of polyps (59 vs. 40%) and adenomas (32 vs. 21%) in NAFLD group. NAFLD was predictive of fnding a polyp on colonoscopy (OR 2.42, P = 0.001) and associated with adenoma (OR 1.95, P = 0.02). aOR for colorectal cancer with varying severity of NAFLD was 1.13 for mild, 1.12 for moderate, and 1.56 for severe (P = 0.007). Cumulative incidence rates of colorectal neoplasm at 3 and 5 years in the NAFLD group were 9.1 and 35.2% vs. 5.0 and 25.3% in non-NAFLD group. NAFLD was associated with increased risk of overall colorectal tumors (aHR 1.31, 95% CI 1.01–1.71), and the development of ≥3 adenomas at the time of surveillance colonoscopy (aHR: 2.49, 95% CI: 1.20–5.20). Higher prevalence of any colorectal neoplasia (38% vs. 28.9%) and advanced colorectal neoplasia (2.8% vs. 1.9%) in NAFLD patients. The aOR in NAFLD patients was 1.1 (95% CI:1.03–1.17) for any colorectal neoplasia and 1.21 (95% CI: 0.99–1.47) for advanced colorectal neoplasia. Higher incidence of colorectal cancer in NAFLD patients (69.4 vs. 34.1 per 100,000 person-years; IRR 2.04; 95% CI 1.3–3.19). NAFLD is an independent risk factor for colorectal adenoma (aOR 1.15; 95%CI 1.02–1.3), advanced adenoma (aOR 1.5; 95% CI 1.12–2.01), and multiple adenomas (aOR 1.32; 95%CI 1.01–1.73). NASH was an independent risk factor for colorectal polyps (OR 2.08; 95% CI 1.12–3.86) and advanced colorectal neoplasm (OR 2.81; 95% CI 1.01–7.87). Higher incidence of colon cancer in NAFLD patients IRR 1.76 (95% CI 1.08–2.8). No signifcant associations between NAFLD and colon cancer (aHR 1.05, 95% CI [0.85–1.28]). The standardized incidence ratio of esophageal cancer was SIR 2.9, 95% CI 0.4–10.3 in the NAFLD group. The prevalence of NAFLD was higher in gastric cancer as compared to the Turkish general population.

20 EXTRAHEPATIC AND HEPATIC CANCERS

Extrahepatic Cancer

Study

Type of Study

Country

Esophageal and gastric

Sun L. M. et al., (2015) [74]

Retrospective cohort

Taiwan

Gastric

Allen A. M. et al. (2019) [73] Simon TG, et al. (2021) [37] Sorensen H. T. et al. (2003) [62] Sun L. M. et al. (2015) [74]

Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort

USA

Pancreas

Kim G. A. et al. (2017) [72]

Retrospective cohort

South Korea

Pancreas

Chang C. F. et al. (2017) [88] Allen A. M. et al. (2019) [73] Simon T. G. et al. (2021) [37] Sorensen H. T. et al. (2003) [62] Sun L.M. et al. (2015) [74]

Case control

Taiwan

Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort

USA

Kidney

Kim G. A. et al. (2017) [72]

Retrospective cohort

South Korea

Kidney/bladder

Simon T.G., et al. (2021) [37] Sorensen H.T. et al. (2003) [62] Kim G.A. et al. (2017) [72]

Retrospective cohort Retrospective cohort Retrospective cohort

Sweden

Simon TG, et al. (2021) [37] Sorensen H.T. et al. (2003) [62] Sun L. M. et al. (2015) [74]

Retrospective cohort Retrospective cohort Retrospective cohort

Sweden

Chu C. H. et al. (2003) [77] Kim G. A. et al. (2017) [72]

Case control

China

Retrospective cohort

South Korea

Kwak M.-S., et al. (2018) [89] Allen AM, et al. (2019) [73] Simon TG, et al. (2021) [37] Sun L. M. et al. (2015) [74]

Case control

South Korea USA

Esophageal and gastric Pancreas Pancreas

Pancreas Pancreas Kidney Kidney

Prostate Prostate

Prostate Breast Breast

Breast Breast

Breast Breast Breast Uterine

Retrospective cohort Retrospective cohort Retrospective cohort

Sweden Denmark Taiwan

Sweden Denmark Taiwan

Denmark South Korea

Denmark Taiwan

Sweden Taiwan

Findings Signifcantly higher incidence of esophageal cancer in the NAFLD with cirrhosis group (aHR 7.25; 95% CI 2.44–21.6) and gastric cancer (aHR 5.5; 95% CI 2.78–10.9). Higher incidence of gastric cancer in NAFLD patients (IRR 2.3, 95% CI 1.3–4.1). No signifcant associations between NAFLD and cancer of the esophagus or stomach (aHR 1.15, 95% CI 0.77–1.71). The standardized incidence ratio of pancreatic cancer was SIR 3, 95% CI 1.3–5.8 in the NAFLD group. Higher crude and adjusted incidence risk of pancreatic cancer in NAFLD with cirrhosis group (HR 4.18; 95% CI 1.67–10.4 and aHR 2.72; 95% CI 0.93–7.95), but the adjusted incidence risk was statistically insignifcant. Higher incidence of pancreatic cancer in NAFLD patients (16 vs. 13.8 per 100,000 person-years) but was statistically insignifcant (IRR 1.16; 95% CI 0.51–2.65). NAFLD was an independent risk factor for pancreatic cancer (OR 2.63, 95% CI 1.24–5.58). Higher incidence of pancreatic cancer in NAFLD patients (IRR 2.1, 95% CI 1.2–3.3) NAFLD patients had signifcantly higher rates of developing pancreatic cancer (aHR, 2.15 [95% CI 1.40–3.30) Standardized incidence ratio of renal cancer in the NAFLD group: SIR 2.7, 95% CI 1.1–5.6. Higher crude and adjusted hazard ratios of urinary system cancer (HR 1.19; 95% CI 0.53–2.66; aHR 1.60; 95% CI 0.69–3.72) in NAFLD with cirrhosis group; however, both were statistically insignifcant. Higher incidence of renal cancer in NAFLD patients (35.6 vs. 20.3 per 100,000 person-years) but was statistically insignifcant (IRR 1.76; 95% CI 0.96–3.22). NAFLD patients had signifcantly higher rates of developing kidney/bladder cancer (aHR, 1.41, 95% CI, 1.07–1.86). The standardized incidence ratio of prostate cancer in the NAFLD group: SIR 1.3, 95% CI 0.5–2.8. Lower incidence of prostate cancer in NAFLD patients (126 vs. 138.9 per 100,000 person-years) but statistically insignifcant (IRR 0.91; 95% CI 0.63–1.31). No signifcant associations between NAFLD and prostate cancer (aHR 0.91, 95% CI 0.77–1.09) Standardized incidence ratio of breast cancer in the NAFLD group: SIR 0.9, 95% CI 0.4–1.7. Higher adjusted incidence risk in the NAFLD with cirrhosis group (aHR 1.23; 95% CI 0.28–5.38) compared to the control group but not statistically signifcant. 45.2% of breast cancer patients vs. 20.3% of the controls had NAFLD (OR 3.23; 95% CI 2.1–5.1, P < 0.0001). Higher incidence of breast cancer in the NAFLD group (181.6 vs. 102.5 per 100,000 person-years) than non-NAFLD group, with IRR 1.77, 95% CI 1.15–2.74. NAFLD was independently associated with breast cancer (OR 1.63, 95% CI 1.01–2.62; P = 0.046). Higher incidence of breast cancer in NAFLD vs. controls, SIR 1.68 (95% CI 1.05–2.76) No signifcant associations between NAFLD and breast cancer (aHR 0.99, 95% CI [0.79–1.24]) Higher crude and adjusted hazard ratio of uterine cancer in the NAFLD with cirrhosis group (HR 2.11; 95% CI 0.67–6.63; and aHR 2.03; 95% CI 0.6–6.8), but both statistically insignifcant.

(Continued) 189

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Table 20.1 (Continued) Extrahepatic Cancer

Study

Type of Study

Country

Uterine

Kim G. A. et al. (2017) [72]

Retrospective cohort

South Korea

Uterine

Allen A. M. et al. (2019) [73] Simon T. G. et al. (2021) [37] Allen A.M. et al. (2019) [73] Simon TG, et al. (2021) [37] Sun L. M. et al. (2015) [74] Allen A.M. et al. (2019) [73] Simon T. G. et al. (2021) [37]

Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort Retrospective cohort

USA

Uterine/cervical/ ovarian Lung Lung Hematologic Hematologic Hematologic

Sweden USA Sweden Taiwan USA Sweden

Findings Higher incidence of uterine/cervical/ovarian cancers in NAFLD patients (48.4 vs. 23.5 per 100,000 person-years) but statistically insignifcant (IRR 2.06; 95% CI 0.86–4.91). Higher incidence of uterine cancer in NAFLD patients (IRR 2.3, 95% CI 1.4–4.1). No signifcant associations between NAFLD and uterine cancer (aHR 0.82, 95% CI 0.61–1.09). No signifcant increase in the incidence rates of lung cancer in NAFLD patients (IRR 1.34, 95% CI 0.87–2.07). No signifcant associations between NAFLD and lung cancer (aHR 1.06, 95%CI 0.82–1.39). Signifcantly higher adjusted incidence of hematologic cancer in the NAFLD with cirrhosis group (aHR 3.12; 95% CI 1.34–7.25). No signifcant increase in the incidence rates of non-Hodgkin lymphoma in NAFLD patients (IRR 0.94, 95% CI 0.52–1.7). NAFLD patients had modest yet signifcantly higher rates of developing hematologic cancers (0.7 vs. 1.0/1,000 PYs; aHR, 1.46, 95% CI, 1.12–1.90).

OR: Odds ratio; CI: Confdence interval; aOR: Adjusted odds ratio; aRR: Adjusted relative risk; aHR: Adjusted hazard ratio; IRR: Incidence rate ratio; SIR: Standardized incidence ratio; HR: Hazard ratio; PY: Person-year.

20.4.2 Prostate CA Large-scale studies have examined the incidence of prostate cancer in patients with NAFLD; however, the results have been inconsistent [71]. A large study from South Korea that included over 10 million males from a national database registry showed a signifcant higher incidence of prostate cancer in patients with NAFLD, defned using surrogate measures (i.e., a hepatic steatosis index 3 36 or fatty liver index 3 60) [71,76]. In a subsequent meta-analysis of 3 large studies (including the aforementioned Korean study), NAFLD was associated with a signifcantly elevated risk of developing prostate cancer [1,72,73,76]. In contrast, other studies failed to show a statistically signifcant association [37], and so whether prostate cancer constitutes a signifcant threat in men with NAFLD remains unclear [71] (see Table 20.1). 20.4.3 Breast CA The association between breast cancer and obesity is well-described [71], and with each 5 kg of weight gain, the risk of breast cancer development increases by 11% [71]. Accordingly, several retrospective studies have shown a similar positive association between NAFLD and breast cancer [71–73,77], the largest of which estimated a 3-fold higher prevalence [77] and a 60–70% increase in incidence [72,73]. In a meta-analysis by Liu et al. that included 4 of these highquality studies, it was concluded that NAFLD patients are more susceptible to the development of breast cancer [1]. However, other, smaller studies have failed to show a signifcant relationship between NAFLD and the incidence or prevalence of breast cancer [62,71,74], and further research is needed to disentangle the risk associated with obesity from any related specifcally to NAFLD (see Table 20.1). 20.4.4 Uterine CA Very few studies have provided evidence for or against an association between NAFLD and uterine cancer, to date [37,71–74] (see Table 20.1). 190

20.4.5 Lung CA Large retrospective cohort studies showed no increased rates of lung cancer in NAFLD patients [37,73] (see Table 20.1). 20.4.6 Hematologic CA A statistically signifcant higher incidence of hematologic cancer was found in a single retrospective cohort study investigating the incidence of cancers in over 2000 NAFLD patients [74]. Similarly, Simon et al. showed that NAFLD patients had modest yet signifcantly higher rates of developing hematologic cancers [37]. The mechanisms that might underpin an association between NAFLD and hematologic cancer development remains undefned, and further research is needed to better characterize these relationships (see Table 20.1). 20.4.7 Is It Time to Change Our Extrahepatic Cancer Screening Strategies for Patients with NAFLD? Given emerging data supporting an increased risk of some extrahepatic cancers in patients with NAFLD, increased clinical awareness is warranted. However, as previously outlined, these data remain preliminary, and further research from large-scale cohorts with well-phenotyped NAFLD and carefully adjudicated clinical outcomes are urgently needed, so that these risks can be confrmed and quantifed at the population level. In the interim, it is important that clinicians ensure that their patients with NAFLD are up-to-date with their recommended cancer surveillance, based on current guidelines. Patients should also be counseled regarding behaviors that may be associated with increased cancer risk [78]. 20.5 CONCLUSION Substantial evidence is accumulating for a role of NAFLD as an independent risk factor for several cancers, particularly hepatic cancers. Data are also accumulating regarding the molecular mechanisms that drive HCC in NAFLD,

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67. VanSaun, M.N., Lee, I.K., Washington, M.K., Matrisian, L., Gorden, D.L. High fat diet induced hepatic steatosis establishes a permissive microenvironment for colorectal metastases and promotes primary dysplasia in a murine model. The American Journal of Pathology. 2009; 175(1): 355–364. 68. Ohashi, K., Wang, Z., Yang, Y.M., Billet, S., Tu, W., Pimienta, M., Cassel, S.L., Pandol, S.J., Lu, S.C., Sutterwala, F.S. NOD-like receptor C4 infammasome regulates the growth of colon cancer liver metastasis in NAFLD. Hepatology. 2019; 70(5): 1582–1599. 69. Hayashi, S., Masuda, H., Shigematsu, M. Liver metastasis rare in colorectal cancer patients with fatty liver. Hepato-gastroenterology. 1997; 44(16): 1069–1075. 70. XI, Z., Fan, Z., Qiu, D., Zeng, M. The relationship between fatty liver disease and liver metastases from colorectal cancer. Chinese Journal of Digestion. 2009; 157–160. 71. Ahmed, O.T., Allen, A.M. Extrahepatic malignancies in nonalcoholic fatty liver disease. Current Hepatology Reports. 2019; 18(4): 455–472. 72. Kim, G.-A., Lee, H.C., Choe, J., Kim, M.J., Lee, M.J., Chang, H.S., Bae, I.Y., Kim, H.K., An, J., Shim, J.H. Association between non-alcoholic fatty liver disease and cancer incidence rate. Journal of Hepatology. 2018; 68(1): 140–146. 73. Allen, A.M., Hicks, S.B., Mara, K.C., Larson, J.J., Therneau, T.M. The risk of incident extrahepatic cancers is higher in non-alcoholic fatty liver disease than obesity—a longitudinal cohort study. Journal of Hepatology. 2019; 71(6): 1229–1236. 74. Sun, L.-M., Lin, M.C., Lin, C.L., Liang, J.A., Jeng, L.B., Kao, C.H., Lu, C.Y. Nonalcoholic cirrhosis increased risk of digestive tract malignancies: a population-based cohort study. Medicine. 2015; 94(49). 75. Watanabe, D., Horiguchi, A., Tasaki, S., Kuroda, K., Sato, A., Asakuma, J., Ito, K., Asano, T., Shinmoto, H. Clinical implication of ectopic liver lipid accumulation in renal cell carcinoma patients without visceral obesity. Scientifc Reports. 2017; 7(1): 1–7. 76. Choi, Y.J., Lee, D.H., Han, K.D., Yoon, H., Shin, C.M., Park, Y.S., Kim, N. Is nonalcoholic fatty liver disease associated with the development of prostate cancer? A nationwide study with 10,516,985 Korean men. PLoS ONE. 2018; 13(9): e0201308. 77. Chu, C.-H., Lin, S.C., Shih, S.C., Kao, C.R., Chou, S.Y. Fatty metamorphosis of the liver in patients with breast cancer: possible associated factors. World Journal of Gastroenterology: WJG. 2003; 9(7): 1618. 78. Wijarnpreecha, K., Aby, E.S., Ahmed, A., Kim, D. Evaluation and management of extrahepatic manifestations of nonalcoholic fatty liver disease. Clinical and Molecular Hepatology. 2021; 27(2): 221. 79. Stadlmayr, A., Aigner, E., Steger, B., Scharinger, L., Lederer, D., Mayr, A., Strasser, M., Brunner, E., Heuberger, A., Hohla, F. Nonalcoholic fatty liver disease: an independent risk factor for colorectal neoplasia. Journal of Internal Medicine. 2011; 270(1): 41–49. 80. Lin, X.-F., Shi, K.Q, You, J., Liu, W.Y., Luo, Y.W., Wu, F.L., Chen, Y.P., Wong, D.K.H., Yuen, M.F., Zheng, M.H. Increased risk of colorectal malignant neoplasm in patients with nonalcoholic fatty liver disease: a large study. Molecular Biology Reports. 2014; 41(5): 2989–2997. 81. Bhatt, B.D., Lukose, T., Siegel, A.B., Brown Jr, R.S., Verna, E.C. Increased risk of colorectal polyps in 193

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86. Cho, Y., Lim, S.K., Joo, S.K., Jeong, D.H., Kim, J.H., Bae, J.M., Park, J.H., Chang, M.S., Lee, D.H., Jung, Y.J. Nonalcoholic steatohepatitis is associated with a higher risk of advanced colorectal neoplasm. Liver International. 2019; 39(9): 1722–1731. 87. Uzel, M., Sahiner, Z., Filik, L. Non-alcoholic fatty liver disease, metabolic syndrome and gastric cancer: Single center experience. Journal of BU ON.: Offcial Journal of the Balkan Union of Oncology. 2015; 20(2): 662–662. 88. Chang, C.-F., Tseng, Y.C., Huang, H.H., Shih, Y.L., Hsieh, T.Y., Lin, H.H. Exploring the relationship between nonalcoholic fatty liver disease and pancreatic cancer by computed tomographic survey. Internal and Emergency Medicine. 2018; 13(2): 191–197. 89. Kwak, M.-S., Yim, J.Y., Yi, A., Chung, G.E., Yang, J.I., Kim, D., Kim, J.S., Noh, D.Y. Nonalcoholic fatty liver disease is associated with breast cancer in nonobese women. Digestive and Liver Disease. 2019; 51(7): 1030–1035.

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21 NAFLD in Children Unique Aspects and Controversies Samar H. Ibrahim and Rohit Kohli

CONTENTS 21.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 21.1.1 Defnition and Nosology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 21.1.2 Epidemiology and Signifcance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 21.2 Current Approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 21.3 Unique Aspects and Gaps in Knowledge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 21.4 Conclusion and Future Directions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 21.1 INTRODUCTION 21.1.1 Defnition and Nosology “A rose by any other name would smell as sweet.” We have all heard if not read this often quoted Shakespearean line of prose. When we speak to fatty liver disease in children, however, nosology and the defnitions (see Table 21.1) it encompasses are both critically important. We are particularly torn by the nosology whereby “nonalcoholic” continues to be used as a defning element for a disease afficting an increasingly large pediatric population worldwide. Each child that is provided this def nition must understand, at some point, the associated stigma inherent with the term “alcoholic.” Given that most will agree that children should not—and the vast majority do not—partake in the consumption of alcohol, a change in the terminology is the need of the hour. We propose the usage of the prefx “Nutrition associated,” thus keeping the acronym “NAFLD/NASH” unchanged. This shift in focus to the crux of the problem, “nutrition,” will allow us to pivot away from the pejorative and negative connotations associated with “nonalcoholic” and hopefully help refocus the efforts of the family on improving the child’s “nutrition.” 21.1.2 Epidemiology and Signifcance The prevalence and incidence of NAFLD in children have been an awakening for pediatric care providers. What was once an obscure diagnosis bringing to

mind a slew of metabolic and inborn errors of metabolism, excess fat in a child’s liver is now, unfortunately, extremely common. The rise of childhood obesity has paralleled the shifts in lifestyles of our children over the past 4 decades. The identifcation of this disorder in the early 1980s (1) was followed by a focus on understanding its epidemiology through population-based cohorts in the early 2000s (2). The epidemiological facts accepted broadly today are listed in Table 21.2. NAFLD in children is truly a signifcant result of the public health tsunami of childhood obesity. We therefore have to acknowledge the limitations of what can be achieved in the four walls of our health care units. With that said, there is clear import to the collaboration of the primary care provider or the gastrointestinal (GI) specialist in making a difference in the lives of these children. Our current suggested approach is outlined in detail here and is encapsulated in an educational video produced by the joint efforts of the Fatty Liver Clinic and the marketing department at Children’s Hospital Los Angeles (https://youtu.be/4CFYMAx5-7E). 21.2 CURRENT APPROACH Patients with NAFLD are often asymptomatic and incidentally identifed by the elevation of liver enzymes or hepatic steatosis on abdominal imaging studies performed for different indications. Although screening for pediatric

Table 21.1: Definitions Fatty liver disease (NAFLD) Fatty liver (NAFL) Steatohepatitis (NASH)

>5% liver steatosis NAFLD − [infammation ± fbrosis] NAFLD + [infammation ± fbrosis]

Table 21.2: Key Points in NAFLD Epidemiology Overall NAFLD rates have been increasing (3). Boys have a higher prevalence of NAFLD (3). Genetics plays a signifcant role in defning the prevalence of the severe form of the disease spectrum, NASH (4). Children of Hispanic and Asian heritage are at increased risk of NASH. Children of African heritage have a lower prevalence of NASH (5). Sugar-sweetened beverage consumption is linked to increased rates of hepatic fbrosis (6)

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DOI: 10.1201/9781003386698-26

21 NAFLD IN CHILDREN

NAFLD is an area of controversy that lacks consensus among different societies (7–9) an ever increasing number of patients are now identifed through screening protocols. We believe that preventive screening in pediatric NAFLD is impactful given the high prevalence, the wide availability of an inexpensive screening tool (alanine aminotransferase [ALT]) and, most importantly, an effective although challenging-to-implement therapy (lifestyle intervention). Furthermore, early intervention in childhood will prevent the onset of end-stage liver disease and improve the patient’s outcome. The North American Society of Pediatric Gastroenterology Hepatology and Nutrition (NASPGHAN) practice guideline recommends screening for NAFLD beginning between ages 9 and 11 years for all obese and overweight children with additional risk factors (e.g., insulin resistance, hyperlipidemia, hypertension, polycystic ovarian syndrome, obstructive sleep apnea, family history of NAFLD) (8). An earlier screening age is warranted in patients with panhypopituitarism given the high prevalence of rapidly progressive fbrosing NASH in this subset of patients (10). The recommended screening test is ALT using sex-specifc upper limits of normal (females, 22 U/L; males, 26 U/L) that represent the 97th percentiles for a healthy lean population, as determined from the National Health and Nutrition Examination Survey (11). It’s important to acknowledge that ALT has some limitations as a screening tool, with advanced fbrosis detected in patients even with mild ALT elevation (12). Screening for gamma-glutamyltransferase (GGT) elevation in addition to ALT will improve the diagnostic accuracy and help identify patients at increased risk of progression. The use of abdominal ultrasound is not recommended as a screening tool for NAFLD (8).

Clinical assessment is of utmost importance in guiding management and is geared to identify: 1. Pediatric NAFLD-causing disorders: Including hypothyroidism (goiter), partial lipodystrophy (abnormal body fat distribution), lysosomal acid lipase (LAL) defciency (xanthelasmas) (13), panhypopituitarism (neurological defcit, short stature and severe obesity); 2. Associated metabolic comorbidities: Hypertension and insulin resistance (assessed by the presence of acanthosis nigricans) 3. Other signs: Of portal hypertension (splenomegaly and spider angioma) and advanced liver disease (sarcopenia, clubbing). Further management is guided by the extent and the duration of the ALT elevation and the response to lifestyle intervention overtime as outlined in Figure 21.1. Indications for referral to the pediatric gastroenterology and hepatology subspecialist are highlighted in Figure 21.1. A comprehensive evaluation is often undertaken by the specialists and aims to rule out other causes of chronic elevation of liver enzymes, to screen for comorbidities, to confrm the diagnosis, and to stage and grade NASH (see Table 21.3). Ultrasound-based shear wave elastography such as FibroScan® is gaining broader use as a noninvasive technique to screen and monitor fbrosis. FibroScan® accurately detects signifcant liver fbrosis. However, the accuracy is reduced with mild liver fbrosis, and the technical failure rate is higher in patients with severe central adiposity (14). The magnetic resonance imaging proton density fat fraction (MRI-PDFF) provides an accurate, validated marker of hepatic steatosis and, when coupled with magnetic

Figure 21.1 Algorithm for the screening and management of pediatric patients with suspected NAFLD * The comprehensive evaluation is outlined in Table 21.3. ** Normal ALT is defned as ≤22 U/L in females and ≤ 26 U/L in males.

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Table 21.3: Compressive Evaluation of Patients with Suspected NAFLD Screening laboratory studies • CBC with differential, AST, GGT, alkaline phosphatase, bilirubin total and direct, albumin, total protein, INR, hemoglobin A1C, and fasting glucose and lipid panel Laboratory evaluation for common cause of chronic elevation of liver enzymes • Autoimmune liver disease: Autoantibody profle (ASMA, ANA, ALKM) • Genetic: Alpha 1 antitrypsin defciency (alpha 1 antitrypsin phenotype); Wilson disease (ceruloplasmin and 24-hour urine copper quantifcation); juvenile hemochromatosis (serum iron, total iron binding capacity, ferritin); lysosomal acid lipase (LAL) defciency (enzymatic assay and genetic study); abetalipoproteinemia (lipoprotein electrophoresis) (19,20) • Viral hepatitis: Hepatitis C antibody, HBsAg and HBsAb • Celiac disease: Tissue transglutaminase antibody (TTG) IgA, total IgA • Hypothyroidism: TSH, FT4 Imaging studies • Abdominal ultrasound: To rule out anatomical abnormalities and to assess for signs of portal hypertension (splenomegaly and nodular liver) • FibroScan®: Considered when available to assess liver fbrosis • MRI-PDFF: When available, considered to measure liver fat and MR elastography to assess liver fbrosis Liver biopsy • To confrm the diagnosis and consider vitamin E • To assess the severity of steatosis, grade the infammation and stage the fbrosis • To rule out alternate liver diseases: Wilson’s disease by copper quantifcation, and autoimmune hepatitis in patients with positive autoantibodies • To support the bariatric surgery indication in select candidates with advanced liver fbrosis

resonance elastography (MRE), serves as a promising noninvasive tool to identify patients with hepatic steatosis and advanced liver fbrosis and to optimize risk stratifcation. However, the MRE does not discriminate between absence of fbrosis and mild fbrosis (15). Furthermore, the high cost and limited availability render it inappropriate as an NAFLD screening tool. Nonetheless, the threedimensional MRE has the potential to assess steatosis (PDFF), infammation (dampening ratio), and fbrosis (elastography) and is emerging as a noninvasive diagnostic and monitoring tool in patients with NASH (16). Indications for a liver biopsy in patients with suspected NAFLD have not been uniformly established, and practice varies. There is a low likelihood that a liver biopsy would change the diagnosis in the majority of pediatric patients suspected to have NAFLD. However, there is merit in identifying a diagnosis of NASH, which opens up the potential for use of vitamin E as a temporary pharmacotherapy (17), details of which are discussed later in this chapter. Hence the decision to pursue a liver biopsy should be made on a case-by-case basis, after discussion of the benefts and risks and shared with the patients and their families. Pediatric patients with NAFLD often have unique histological features that include portal infammation and fbrosis in the absence of hepatocellular ballooning; this pattern is referred to as “type 2” NAFLD, while the pattern typically seen in adults is referred to as “type 1” NAFLD (18). The management of pediatric NAFLD is geared to improving the outcome, achieving NASH resolution and fbrosis regression. The treatment is a three-pronged approach with lifestyle intervention, medical therapy and bariatric surgery. Lifestyle intervention with diet and exercise to achieve 7–10% weight loss remains the cornerstone in the management of NASH. In a relatively recent systemic review and meta-analysis of 19 studies that included 923 patients, aerobic exercise and diet were the most used interventions, 198

and in two studies aerobic exercise was the only intervention. The age of participants ranged from 6 to 18 years. The diet employed was normo-caloric ranging from 1,300 to 1,900 Kcal/day (50–65% from carbohydrates, 10–30% from fat and 12–20% from protein). All studies assessed aerobic exercise, ranging from once a week to daily workouts of 60 minutes on average, mostly 3–7 times per week. The duration of intervention ranged between 4 and 52 weeks. lifestyle changes to treat NAFLD in this study showed signifcant improvements in BMI, ALT levels and hepatic steatosis (21). Emerging studies suggest that a low-glycemic, lowadded-sugar diet (22) is benefcial in patients with NAFLD when adopted by the whole family. Recently, there has been increased interest in employing the Mediterranean diet in NAFLD patients due to the high-fber, polyunsaturated-fatty-acids, and antioxidants content (23). Adult, randomized clinical trials using the Mediterranean diet showed signifcant weight loss, greater improvement in ALT and liver stiffness and higher adherence (24). In addition, training with aerobic plus resistance exercise led to greater changes in ALT and greater resolution of hepatic steatosis than aerobic training alone (25). Hence physical activity is essential in the management of NASH and may be benefcial in the absence of weight loss supporting the importance of limiting screen time to no more than 2 hours a day. The frequency of the visit and the accountability system, as well as the management by a multidisciplinary team, are essential in achieving and maintaining weight loss in patients with NAFLD (26). Children and adolescents with NAFLD should be counseled that alcohol consumption may exacerbate NAFLD. Assuring immunity after vaccination for hepatitis B and hepatitis A is important in NAFLD patients to eliminate the risk of vaccine preventable viral hepatitis. Medications approved for weight loss in adolescents and adults include liraglutide and orlistat, but these have not been studied as NAFLD therapy in children. Although

21 NAFLD IN CHILDREN

pediatric NAFLD lacks regulatory agency-approved therapy, here we will focus on medical agents in clinical use in pediatrics rather than in the pipeline. The Treatment of NAFLD in Children (TONIC) trial evaluating vitamin E or metformin in 173 children ages 8 to 17 years with biopsy-proven NAFLD showed that daily vitamin E (800 IU) enhanced NASH resolution when compared to placebo without signifcant change in ALT (17). The same study did not show effcacy of metformin therapy. Hence, we recommend vitamin E in patients with biopsy-proven NASH. The potential for n-3 PUFA supplementation in reducing hepatic steatosis and blood triglyceride level in children with NAFLD is well-established (27). However, there was no improvement in ALT or components of the metabolic syndrome to recommend supplementation with n-3 PUFA as a treatment of NAFLD (28). Although small studies in pediatrics using probiotic have shown improvement of ALT and hepatic steatosis over the short period of administration (29), the benefcial role of probiotic as NAFLD therapy has not been established, and additional randomized controlled trial are required (30) before probiotics are recommended. Selection criteria for bariatric surgery include a BMI 35–39 or 120% of the 95th percentile with comorbidities (type 2 diabetes mellitus, obstructive sleep apnea, advanced NASH, pseudotumor cerebri, Blount’s disease, slipped capital femoral epiphysis, gastroesophageal refux, and hypertension) or a BMI ≥40 or 140% of 95th percentile without any additional comorbidities requirement to qualify for bariatric surgery. The bariatric surgery evaluation includes a multidisciplinary team assessment of ability and motivation to adhere to pre- and postoperative treatment recommendations, including micronutrient supplements (31). Operations used in adolescents include restrictive surgeries (when the stomach volume is surgically reduced) and malabsorptive (when the small bowel absorptive area is bypassed). Sleeve gastrectomy (SG) and gastric band (GB) are restrictive surgeries while Rouxen-Y gastric bypass (RYGB) is malabsorptive and restrictive. A comparative study that included adolescents with RYGB (n = 177), SG (n = 306) and laparoscopic adjustable gastric banding (n = 61) showed that since 2005 the use of the SG bariatric surgery approach has gradually increased, while the RYGB and GB approaches have declined.

Furthermore, the mean BMI changes were respectively −31% for RYGB, −28% for SG and −10% for GB at 1 year. Similar trends were seen at 3 years, suggesting that laparoscopic adjustable gastric banding was signifcantly less effective for BMI reduction than SG and RYGB (32). Bariatric surgery in adolescents is associated with signifcant improvements in weight loss, diabetes, prediabetes, dyslipidemia, hypertension and kidney disfunction, and weight-related quality of life at 3 years after the procedure. Risks associated with surgery included low ferritin and the need for additional complication-related abdominal procedures (33). Endoscopic bariatric surgery has been established in adults with some emerging literature supporting safety and effcacy of endoscopically an inserted gastric balloon in adolescents (34) (Figure 21.2). This is a potential therapeutic strategy that puts together the benefts of weight loss surgery sans the surgery! 21.3 UNIQUE ASPECTS AND GAPS IN KNOWLEDGE The natural history and long-term risk of mortality in children and young adults with biopsy-confrmed NAFLD is poorly defned. Emerging data suggest progression of NAFLD in patients receiving lifestyle intervention (35), as well as higher rates of cancer, liver- and cardiometabolicspecifc mortality (36). In a nationwide, matched cohort of all Swedish children and young adults (≤25 years) with biopsy-confrmed NAFLD, the 20-year absolute risk of overall mortality was 7.7 % among NAFLD vs. 1.1% in the general population (36). While screening tools for NAFLD are evolving, the precision of some including the FibroScan® requires further validation, and MR elastography is more available than before but is still not very widely available. Therefore, the role of noninvasive monitoring is crucial in the pediatric population to abrogate the risks of a liver biopsy performed usually under anesthesia in the pediatric age group. Furthermore, pediatric NAFLD is a heterogeneous disease infuenced by age, sex, genetic variants, metabolic comorbidities and the microbiota composition. The heterogeneous nature of NAFLD results from a different contributions of numerous pathogenic mechanisms manifesting as multiple disease phenotypes with different natural history, prognosis and response to therapy (37).

Figure 21.2 Endoscopically inserted gastric balloon, appropriate position confrmed by X-ray (Courtesy of Dr. Imad Absah, Mayo Clinic.) 199

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The role of disease modifer in the severity of NASH is unraveling. A well-described example is alpha-1 antitrypsin defciency, where the heterozygotes (MZ) genotype may contribute to the severity of NAFLD and the need of liver transplantation (38). In addition, intestinal dysbiosis plays a critical role in the development of pediatric NAFLD but is not clinically used in the diagnosis and management. A subset of patients with rapidly progressive NASH associated with panhypopituitarism may require liver transplantation in adolescence or young adulthood and are at high risk of recurrence in the graft (39,40). All these factors account for the modest response to therapies applied on a diverse patient population without stratifcation. Adding to the challenges of managing pediatric NAFLD is that weight loss and maintenance are not achieved without family-based, patient-centered intensive interventions. Increased awareness and advocacy for the development of public health policies that support healthy school meals and the design of cities that incorporate space for children’s outdoor activities are essential in the journey to prevent and treat pediatric NAFLD. 21.4 CONCLUSION AND FUTURE DIRECTIONS In summation, we are faced with a challenge that is society’s own making. The genes that predispose us to the severe form of this disease, NASH, have been in existence for millennia, but the change in our nutritional environment that has been brought to bear on these genes is no more than a few decades in the making. We liken this challenge to our dependence as a society on fossil fuels. There is a way out through green energy for the latter, and there is a way out by reversing our current mal-lifestyle for the former. While we await the population-based public health endeavors to bear fruit, we will still have to take care of the children with NASH and their progressive fbrosis leading to end-stage liver disease and the need for liver transplant in early adulthood. To those children we need to deliver safe and effective medications and/or weight loss strategies. These may even include, for the very extreme scenarios, weight loss surgical techniques that are currently exclusive to adults, such as endoscopic bariatric surgery or devices. These medications and procedures are, of course, not ready for “prime time” yet; however, we should not be surprised to see them brought up in our pediatric practices and discussed by families as therapeutic options for their children who may unfortunately have NASH. When that does happen, we better be prepared to discuss the smell of these new roses! REFERENCES 1. Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo clinic experiences with a hitherto unnamed disease. Mayo Clin Proc. 1980;55(7):434–438. Epub 1980/07/01. PubMed PMID: 7382552. 2. Schwimmer JB, Deutsch R, Kahen T, Lavine JE, Stanley C, Behling C. Prevalence of fatty liver in children and adolescents. Pediatrics. 2006;118(4):1388–1393. Epub 2006/10/04. doi:10.1542/peds.2006-1212. PubMed PMID: 17015527. 3. Zhang X, Wu M, Liu Z, Yuan H, Wu X, Shi T, et al. Increasing prevalence of NAFLD/NASH among children, adolescents and young adults from 1990 to 2017: A population-based observational study. BMJ Open. 2021;11(5):e042843. Epub 2021/05/06. doi:10.1136/

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33. Inge TH, Courcoulas AP, Jenkins TM, Michalsky MP, Helmrath MA, Brandt ML, et al. Weight loss and health status 3 years after bariatric surgery in adolescents. N Engl J Med. 2016;374(2):113–123. Epub 2015/11/07. doi:10.1056/NEJMoa1506699. PubMed PMID: 26544725; PubMed Central PMCID: PMCPMC4810437. 34. De Peppo F, Caccamo R, Adorisio O, Ceriati E, Marchetti P, Contursi A, et al. The Obalon swallowable intragastric balloon in pediatric and adolescent morbid obesity. Endosc Int Open. 2017;5(1):E59–E63. Epub 2017/02/10. doi:10.1055/s-0042-120413. PubMed PMID: 28180149; PubMed Central PMCID: PMCPMC5283171. 35. Xanthakos SA, Lavine JE, Yates KP, Schwimmer JB, Molleston JP, Rosenthal P, et al. Progression of fatty liver disease in children receiving standard of care lifestyle advice. Gastroenterology. 2020;159(5):1731–1751 e10. Epub 2020/07/28. doi:10.1053/j.gastro.2020.07.034. PubMed PMID: 32712103; PubMed Central PMCID: PMCPMC7680281. 36. Simon TG, Roelstraete B, Hartjes K, Shah U, Khalili H, Arnell H, et al. Non-alcoholic fatty liver disease in children and young adults is associated with increased long-term mortality. J Hepatol. 2021;75(5):1034–1041. Epub 2021/07/06. doi:10.1016/j.jhep.2021.06.034. PubMed PMID: 34224779; PubMed Central PMCID: PMCPMC8530955.

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22 NAFLD IN HIV PATIENTS

22 NAFLD in HIV Patients Giada Sebastiani

CONTENTS 22.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 22.2 Defnition of NAFLD in HIV. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 22.3 Pathogenesis of NAFLD in the Context of HIV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 22.3.1 Classic Pathogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 22.3.2 Pathogenic Factors Specifc to HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 22.4 Epidemiology of NAFLD in People with HIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 22.5 Natural History of HIV-Associated NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 22.6 Diagnostic Tools for NAFLD, Liver Fibrosis and Esophageal Varices in HIV Mono-Infected Patients . . . . . . . . . . 209 22.7 Models of Care for HIV-Associated NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 22.8 Treatment of HIV-Associated NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 22.8.1 Lifestyle Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 22.8.2 ART-Related Interventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 22.8.3 Pharmacologic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 22.8.4 Bariatric Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 22.9 Conclusion/Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210 22.10 Confict of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 22.11 Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Key Points ■

Aging-related comorbidities, including liver disease, represent the main drivers of morbidity and mortality in people with HIV (PWH). This trend is driven by the successful implementation of effective antiretroviral therapy (ART), with PWH now reaching a life expectancy similar to that of the general population.

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Nonalcoholic fatty liver disease (NAFLD) is a frequent aging-related comorbidity, affecting 35% of HIV monoinfected patients.

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PWH are at higher risk not only of NAFLD but also of NASH and associated liver fbrosis. Multiple pathogenic mechanisms may be involved, including excess metabolic comorbidities, hepatotoxic effect of lifelong ART and immunoactivation due to chronic HIV infection.

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Noninvasive diagnostic tests, such as serum biomarkers and elastography, may help case fnding of PWH with NAFLD-related fbrosis. This will improve risk stratifcation and enhancement of clinical management decisions, including prompt initiation of interventions such as lifestyle changes and a few pharmacologic interventions, as well as surveillance for hepatocellular carcinoma and esophageal varices.

22.1 INTRODUCTION HIV continues to be a major global public health issue. In 2018, there were approximately 37.9 million people with HIV (PWH) worldwide (1). The advent of combination antiretroviral therapy (ART) has considerably improved the health of PWH: 50% of PWH in North America are now over 50 years old (2). As a consequence, the focus in the management of PWH is shifting to chronic noninfectious comorbidities as both chronic HIV infection itself and long-term ART may affect the trajectory of aging-related conditions (3). Nowadays, mortality from liver disease is higher than that from cardiovascular diseases and second only to AIDS-related mortality (4). Over the last decade, DOI: 10.1201/9781003386698-27

the proportion of deaths attributed to liver-related causes has increased between 8- and 10-fold in the post-ART era while AIDS-related mortality has fallen more than 90-fold (5, 6). While coinfection with hepatitis B (HBV) and C (HCV) viruses is believed to have driven this trend in the past, risk factors unique to this population, combined with frequent metabolic comorbidities, may contribute to nonalcoholic fatty liver disease (NAFLD) in HIV mono-infected patients, who represent 86–89% of PWH (7). 22.2 DEFINITION OF NAFLD IN HIV NAFLD is an umbrella term that encompasses a spectrum of clinical and pathologic features characterized by a fatty overload involving over 5% of the liver weight in the absence of other causes of liver disease. Nonalcoholic fatty liver or simple steatosis can evolve to nonalcoholic steatohepatitis (NASH), signifcant scarring (fbrosis) and liver cirrhosis, eventually resulting in end-stage complications (8). Metabolic-associated fatty liver disease (MAFLD) is a novel concept proposed in 2020 aiming to replace the term NAFLD (9). Unlike NAFLD, MAFLD is not a diagnosis of exclusion of other liver diseases, such as excessive alcohol consumption or viral hepatitis. MAFLD is a positive diagnosis done in patients when they have both hepatic steatosis and any of the following three metabolic conditions: overweight/obesity, diabetes mellitus or evidence of metabolic dysregulation in lean individuals. This novel concept has also been proposed in HIV, especially considering that lean NAFLD seems more prevalent and severe in the setting of HIV infection (10,11). However further data on its natural history and utility in clinical practice are needed in the setting of HIV infection (12). 22.3 PATHOGENESIS OF NAFLD IN THE CONTEXT OF HIV INFECTION 22.3.1 Classic Pathogenic Factors The pathogenesis of NAFLD in PWH encompasses multiple complex mechanisms, including frequent metabolic 203

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pathogenic factors and risk factors specifc to HIV infection. These mechanisms are only partially understood and include comorbid metabolic conditions, direct viral effects and adverse effects of ART (see Figure 22.1). In HIVnegative NAFLD, insulin resistance represents the major driver of disease pathogenesis. Moreover, any element constituting the metabolic syndrome, such as obesity, type 2 diabetes mellitus (T2DM), hypertension or dyslipidemia, is linked to progression of NAFLD, and 85% of patients with NAFLD have at least one such condition (13). These classic components of the metabolic syndrome are more frequent in PWH. Diabetes is four times more prevalent in PWH compared to HIV-negative men. A longitudinal study with a median follow-up of 4 years found a cumulative incidence of T2DM of 10% in PWH, compared to 3% in uninfected controls (14). A meta-analysis of 44 studies reported a pooled incidence rate of overt diabetes and prediabetes at 13.7 per 1000 person-years (PY) of followup (95% confdence interval [CI] 13–20; I = 98.1%) among 396,496 PY and 125 per 1000 PY (95% CI 0–123; I = 99.4%) among 1,532 PY, respectively (15). In PWH, dyslipidemia is a complex condition due to multiple contributing factors including the HIV virus itself, individual genetic characteristics and ART-induced metabolic changes (16). There is evidence of an abnormal HDL cholesterol metabolism in PWH compared with uninfected persons. The prevalence of dyslipidemia in PWH ranges from 35 to 63% (17,18). Among ART regimens, protease inhibitors (PIs) generally increase LDL cholesterol and triglycerides, most notably when paired with ritonavir as a pharmacological booster (16). A study of 13,632 adults reported an incidence rate of dyslipidemia higher in ART-treated compared to ARTnaïve and matched non-HIV groups (24.55 per 1,000 PY vs. 14.32 vs. 23.23, respectively). Multivariable analysis suggested a higher risk of dyslipidemia in the ART-treated HIV-infected group (adjusted hazard ratio [aHR],=,1.18; 95% CI 1.07–1.30] and a lower risk in the ART-naïve HIVinfected group (aHR = 0.66; 95% CI 0.53–0.82) compared to the control non-HIV-infected group (19). Hypertension is also very common in PWH. A recent systematic review and meta-analysis including 194 studies (396,776 PWH from 61 countries) reported a global prevalence of hypertension at 23.6% (95% CI 21.6–25.5). The prevalence was higher in high-income countries and in PWH taking ART (20). Finally, the increased prevalence of NAFLD in PWH is paralleled by the concomitant increase in overweight and obesity rate (21). Interestingly, lean NAFLD, def ned as NAFLD affecting patients with body mass index (BMI) less than 25 kg/m2, seems more frequent in the setting of HIV infection. Indeed, NAFLD affects 1 in 4 lean PWH, representing 35% of all PWH with NAFLD. Lean PWH with NAFLD have also more metabolic derangements, such as higher triglyceride and alanine aminotransferase (ALT) levels, and lower HDL levels than lean patients without NAFLD. Finally, they also have longer duration of HIV infection and higher CD4 lymphocyte counts and are more likely to be virally suppressed (22). Besides insulin resistance, the pathophysiology of NAFLD is infuenced by multiple factors (environmental and genetics) in a multiple parallel-hit model, in which oxidative stress may play a primary role. The homeostasis of fat and energy in hepatic cells is regulated by mitochondrial activities, including beta-oxidation of free fatty acids, electron transfer and production of adenosine triphosphate and reactive oxygen species (ROS) (23). Mitochondrial abnormalities

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alter the balance between pro-oxidant and antioxidant mechanisms, leading to an increase of fatty acids as a result of the blockade of beta-oxidation and the consequent production of ROS (23). A decreased activity of several antioxidant enzymes is also observed. Glutathione peroxidase activity is reduced in NAFLD, and mitochondrial cytochrome P450 2E1, a potential direct source of ROS, has an increased activity in NASH patients. Some cytochrome P450 2E1 polymorphisms have been shown to be associated with the development of NASH in obese, nondiabetic subjects (23). Overall, oxidative stress triggers production of infammatory cytokines (such as interleukin 6 and cytokeratin 18) and stimulates fbrogenesis and cell death (8) in NAFLD. Of note, PWH have high levels of markers of oxidative stress (24). Genetic predisposition also contributes to NAFLD pathogenesis. A genomewide association scan of sequence variations (n = 9,229) in a multiethnic population identifed an allele variant of the patatin-like phospholipase domain containing 3 (PNPLA3) gene (rs738409; I148M) as strongly linked to more hepatic infammation and fat content deposition (25). In the Multicenter AIDS Cohort Study, PNPLA3 (rs738409) non-CC genotype was associated with a higher prevalence of fatty liver (odds ratio 3.30, 95% CI 1.66–6.57), although this was not confrmed by a subsequent study (26,27). Gut microbiota has emerged as a potential player in the pathogenesis of NASH. In a study of 81 PWH, Yanavich and colleagues found that those with steatosis or fbrosis had distinct microbial profles, specifcally PWH with steatosis had depletions of Akkermansia muciniphila and Bacteroides dorei and enrichment of Prevotella copri, Finegoldia magna and Ruminococcus bromii, while PWH with fbrosis had depletions of Bacteroides stercoris and Parabacteroides distasonis and enrichment of Sneathia sanguinegens (28). Finally, monocyte/macrophage activation (soluble CD163 and CD14) may affect development of NAFLD and fbrosis, suggesting a Kupffer cell activation in the development of liver fbrosis. Soluble CD163 and CD14 have been associated with immune dysfunction, all-cause mortality and liver fbrosis in PWH (29, 30). 22.3.2 Pathogenic Factors Specifc to HIV Factors unique to PWH may further contribute to the increased frequency and severity of NAFLD. Direct viral effects may contribute to NAFLD. Chronic HIV infection and associated infammation may lead to immuneactivating and pro-apoptotic effects of HIV on hepatocytes, including low-level HIV replication in hepatocytes inducing liver fbrosis (31,32). Indeed, HIV viremia from ART interruptions is an independent risk factors for chronic elevated transaminases (33). Depletion of CD4+ lymphocytes in the gut causes disruption of the gut epithelial barrier, facilitating microbial translocation into the portal and systemic translocation. This promotes liver fbrosis by activation of hepatic Kupffer cells and induction of proinfammatory cytokines (34). HIV-induced mitochondrial dysfunction results in the production of ROS, which causes oxidative stress increasing fat accumulation in hepatocytes (35). Certain ART regimens may also contribute to the pathogenesis of NAFLD. Chronic elevation of ALT is noted in 20–30% of PWH on ART and is associated with histologic abnormalities, including NASH and fbrosis, in up to 60% of cases (31,36). Older nucleoside reverse transcriptase inhibitors (NRTIs), particularly zidovudine, stavudine and didanosine, can induce mitochondrial

22 NAFLD IN HIV PATIENTS

Figure 22.1

Adverse effects of ART

damage leading to impaired fatty acid oxidation and microvesicular steatosis. Although these drugs are no longer recommended, associated insults on the liver persist after discontinuation and may be irreversible (16,37,38). PIs increase central adiposity, decrease hepatic clearance of very-low-density lipoproteins, increase hepatic triglyceride production and potentially contribute to NAFLD (39). Use of ritonavir-boosted PIs (darunavir, indinavir, lopinavir) is commonly associated with elevation in transaminases and direct hepatocyte stress (40). Although less frequent with more modern ART regimens, lipodystrophy is also a potential pathogenetic contributor to NAFLD in PWH. Lipodystrophy is a constellation of body composition and metabolic alterations characterized by a pathological accumulation of adipose tissue in the abdominal region, insulin resistance and dyslipidemia (41). Lipodystrophy may occur in up to 80% of PWH treated with old ART regimens, particularly PIs, and persists after their discontinuation. NAFLD may coexist as integral part of this clinical entity. ART has been associated with weight gain and with metabolic consequences still not known (42). In a US study, nearly half of PWH were obese at the time of starting ART, and in the following 2 years, 20% moved up to a higher BMI category (43). Achhra and colleagues showed that weight gain in the frst year after initiation of ART was associated with increased risk of cardiovascular disease and diabetes (44). Despite integrase inhibitors being associated with a safer metabolic profle and less frequent dyslipidemia, recent data suggest increased weight gain, especially in Black African women (45). In a single-center, prospective study of 301 PWH followed for a median of 41.8 months, participants who received integrase inhibitors and tenofovir alafenamide-based ART demonstrated a signifcantly faster steatosis development or progression by transient elastography (TE) with associated controlled attenuation parameter (CAP) (46). Overall, the pathogenesis of NAFLD in the setting of HIV infection is complex

and only partly understood, due to both HIV itself and the long-lasting exposure to ART, combined with frequent metabolic comorbidities. 22.4 EPIDEMIOLOGY OF NAFLD IN PEOPLE WITH HIV With longer life expectancy and declining AIDSassociated mortality, non-AIDS-related comorbidities have emerged as major diagnostic and treatment concerns in PWH. Chronic liver disease is now the second most common cause of non-AIDS-related mortality in PWH (4). Results of a US study of 47,062 PWH from 2006 to 2016 showed that 22% had some form of liver disease (47). The increased prevalence of liver disease is multifactorial, but an important component is likely attributable to NAFLD. The prevalence of NAFLD among PWH ranges widely from 13 to 65%, likely due to differences in study population characteristics and diagnostic tools adopted (Table 22.1) (38,48–52). For instance, the proportion of PWH with elevated ALT, distribution of sex and ethnicity, as well as frequency of metabolic comorbidities and HIV characteristics—particularly type and duration of ART exposure and HIV infection—affect this reported prevalence. Older studies included mostly PWH with chronic elevation of liver transaminases, selected to undergo liver biopsy. In those studies, the prevalence of NAFLD was as high as 65% (36,53). Most recently, studies employing noninvasive diagnostic tools in consecutive PWH without viral hepatitis coinfection reported a prevalence ranging between 30 and 40% (54–56). Histologic studies reporting the prevalence of NASH in HIV mono-infection mostly included patients with elevated ALT, thus introducing a selection bias (57). A study from UK including 97 HIV mono-infected patients reported a prevalence of 8.2% (58). In a study of 55 patients with elevated ALT, Crum– Cianfone et al. found a prevalence of NASH at 7.3% (59). The largest histology-based study including 116 PWH 205

206

Table 22.1a: Prevalence of NAFLD/NASH and Significant Liver Fibrosis/Cirrhosis in HIV Mono-Infected Patients  

Design/ Country

N

Diagnostic Method

Benmassaoud Prospective/ (63) Canada

202

CrumCianfone (59)

216 (Biopsy US 41 (IQR n = 55) (liver biopsy 30–46) in a subgroup) 255 CT scan 48 (range 19–74)

Prospective/ USA

Guaraldi (38) Prospective/ Italy

Prospective/ France

Lemoine(61)

Prospective/ France Lombardi(51) Retrospective/ UK Lombardi Retrospective/ (66) UK Lui (50) Prospective/ China Macias (49) Prospective/ Spain Mohr (56) Prospective/ Germany

Morse (53)

53.8+ 10.5

77.7

0

NA

96.2

12.5

72.4

97

30

Liver biopsy 46 (range 31–67)

14

Liver biopsy 43.5 (range 86 31–58) Liver biopsy 47.5 + 8.5 91.7

20 (out of 156) 125 US/TE 80 505

H-MRS/ CK−18/TE CAP

289

CAP/TE

Prospective/ USA

62

Nishijima (48) Prospective/ Japan

435

39.6+10.3

13.4

75

26.0+4.1

5.1

100

0

23.7+3.4

12.2

28

0

23.0+3.1

NA

0

23.0+3.4

0

NA

91

6.5

NA

NA

53.9/11.4 10.9/4.5

14 (IQR 6–20)

NA

31/7.3

12.3 + 5.0

NA

36.9/NA NA

100

13 (IQR 9–15)

NA

60/53.3

NA

100

10.6 (median)

NA

57.1/57.1 28.6

AST/ALT ratio, male sex, waist circumference, NRTI exposure NASH: ↑TG, hyperglycemia, HOMA-IR NA

11

100

14 (IQR 2–30)

11 (IQR 0–26) 3 (IQR 0–17) NA

65/NA

NA/5

NA

55/NA

17.6/NA

28.7/NA 13.8//5

Male sex, age, HOMA-IR, GGT ↑TG

NA

40/NA

BMI

24.6+2.9

5.6

16.8

6 (IQR 0–26)

53.9 + 11.2 92.5

1

23.6 + 3.9

48.8

NA

46 (IQR 41–49) 45 (IQR 20–75)

69

11

4.4

NA

78

NA

23.2 (IQR 20.9–26) 24 (range 16–41)

4

NA

Liver biopsy 50 (range 17–67)

94

0

27.6 (range 9.7 15.3–47.1)

100

17.5 (range 2.3–27.8)

US

93

0

22.1 (IQR 5.1 20.2–24.9)

NA

NA

10 (range 35–50)

NAFLD Predictors

8 (IQR 4–13) NA 8 (range 0–29)

6 (range 0–23)

3.6

20

NA

40.8/NA NA

12.9 (range 72.6/54.8 19.4 1.7–22.8)

1.4 (range 31/NA 0–5.6)

NA

NASH: HOMA-IR, ↑ALT NA

BMI, haemoglobin glycosylated, TG NASH: obesity, insulin resistance, PNPLA3 BMI, dyslipidaemia, AST/ALT ratio

SECTION V: NAFLD IN SPECIAL POPULATIONS

Ingiliz (36)

CAP/TE, CK−18

Age Male Alcohol BMI Diabetes ALT Duration HIV Time on NAFLD/ –> F2 Fibrosis/ (years) (%) Excess (Kg/m2) (%) Elevation Infection ART NASH cirrhosis (%) (%) (%) (years) (years) (%)

Perazzo (68)

Prospective/ Brazil

Prat (58)

Retrospective/ 97 UK Prospective/ 122 USA Prospective/ 14 USA

Price (52) Sterling (57)

Vodkin (65)

Prospective/ USA VuilleProspective/ Lessard (54) Canada

395

33 300

CAP/TE

45 (IQR 35.52)

40

23

25.7 (IQR 10 23.2–29.4)

Liver biopsy 47+10

93

20

27+6

H-MRS

51 (IQR 47–57) Liver biopsy 45+10

53

14

71

0

26 (IQR 24–30) 29.9+7.4

Liver biopsy 44.8+9.8

78.8

0

CAP/TE

43.3

0

50

10 (6–16)

7 (range 4–14)

35/NA

9/4.9

100

10.5+9.3

8.3+7.2

28.7/8.2

20

NA

NA

0

100

NA

7.9 (IQR 28/NA NA/2.5 3.3–12.5) NA 64.3/28.6 14.3

29.8+6.0

18.2

NA

NA

NA

100/63.6

33.3/6

NA

11.3

NA

NA

NA

48/NA

15/2.3

11 8.2

3

Central obesity, diabetes, dyslipidemia, metabolic syndrome NA HIV RNA, HOMA-IR NAFLD: ↑GGT NASH: HOMA-IR NASH: HIV duration BMI, ↑ALT

ALT: Alanine aminotransferase; ART: Antiretroviral therapy; BMI: Body mass index; CAP: Controlled attenuation parameter; CK-18: Cytokeratin 18; CT: Computed tomography; HIV: Human immunodefciency virus; IQR: Interquartile range; GGT: Gamma glutamyl transferase; H-MRS: Proton magnetic resonance spectroscopy; HOMA-IR: Homeostatic model assessment of insulin resistance; NA: Not available; NAFLD: Nonalcoholic fatty liver disease; NASH: Nonalcoholic steatohepatitis; NRTI: Nucleoside reversal transcriptase inhibitors; PNPLA3: Patatin-like phospholipase domain-containing protein 3; TE: Transient elastography; TG: Triglycerides; US: Ultrasound.

22 NAFLD IN HIV PATIENTS

Legend: Continuous variables are expressed as mean + standard deviation or median (interquartile range or range), and categorical variables are presented as percentages. Signifcant liver fbrosis is defned as stage >F2 or equivalent.

207

208

Table 22.1b: Incidence of NAFLD and/or Significant/Advanced Liver Fibrosis in HIV-Infected Patients  

Design/ Country

N

Age (years)

Duration HIV Diagnostic Method Infection (years)

Duration Follow-up

210

44.3 +9.7

8.89 + 5.3

TE>7.2 kPa

Sebastiani (41)

Prospective/ Canada 2015

796

43.5 (IQR 36–49.7)

6.3 (IQR 1.7–13.3)

Pembroke (40)

Prospective/ Canada 2017

313 50 (43–54) 15 (IQR 8–22) (HIV and HIV/HCV)

LallukkaBruck (42)

Retrospectiveprospective/ Finland 2019

42

Hepatic steatosis 4.9 years (IQR index >36; FIB-4 2.2–6.4) >3.25 CAP > 248 dB/m or 15.4 months transition to > 292 (IQR TE > 7.1 kPa or 8.5–23.0) transition to >12.5 H-MRS, LFAT > 5.56% 15.7 years TE > 8.7kPa/ (range MRE>3.62 12.3–16.4)

41.9 + 1.3

23.5 + 0.7

18 months (IQR 12–26)

Liver Fibrosis

Predictor of Progression Steatosis

NA

10.9% (end of NA follow-up)

6.9 per 100 PY (95% CI, 5.9–7.9) 37.8 per 100 PY (95% CI, 29.2–49.0)

0.9 per 100 PY (95% CI, 0.6–1.3) 12.7 per 100 PY (95% CI, 9.5–17.1)

Prevalence 9.5% (end of baseline vs. end follow-up) of follow-up: 35% vs. 32%

Fibrosis

No association with ART drugs and length of exposure to drugs Black ethnicity Hyperglycemia Lower level of Lower level of albumin albumin NA In HIV mono-infected: HIV duration, any grade of NAFL; In HIV/ HCV: ↑ALT, HCV RNA NA NA

Legend: Continuous variables are expressed as mean + standard deviation or median (interquartile range or range), and categorical variables are presented as percentages. ALT: Alanine aminotransferase; ART: Antiretroviral therapy; CAP: Controlled attenuation parameter; CI: Confdence interval; HIV: Human immunodefciency virus; HCV: Hepatitis C virus; H-MRS: Proton magnetic resonance spectroscopy; IQR: Interquartile range; LFAT: Liver fat; MRE: Magnetic resonance elastography; NA: Not available; NAFLD: Nnonalcoholic fatty liver disease; PY: Person-years; TE: Transient elastography.

SECTION V: NAFLD IN SPECIAL POPULATIONS

RiveroProspective/ Juarez (43) Spain 2013

NAFLD

22 NAFLD IN HIV PATIENTS

with abnormal liver transaminases found a prevalence of NASH at 49% (60). Other studies found even higher prevalences of NASH, ranging from 53 to 64% (36,53,61,62). A case-control study from US found that, compared to age- and sex-matched HIV-negative NAFLD, patients with HIV-associated NAFLD had signifcantly higher rates of steatohepatitis (37 vs. 63%) and more hepatocyte injury (62). A Canadian study from our team used the biomarker of hepatocyte apoptosis cytokeratin 18 to screen 202 consecutive PWH for NASH (63). The prevalence of NASH was at 11.4%, confrmed in a subgroup by available liver histology. Increased severity of liver disease in PWH, as indicated by higher prevalence of signifcant liver fbrosis and cirrhosis, has also been reported. The prevalence of signifcant liver fbrosis due to NAFLD in HIV monoinfected ranges widely from 3.6 to 35.7% (64,65). Across studies employing TE, this prevalence ranges between 9 and 18%(66–68). Similar results were obtained by Guaraldi et al. when the biomarker FIB-4 was used (38). A study employing AST-to-platelet ratio index (APRI) found a prevalence of liver cirrhosis at 2.5% (52). In a metaanalysis of 10 studies in PWH, the prevalence of NAFLD, biopsy-proven NASH, and signifcant liver fbrosis were 35, 42 and 22%, respectively, all higher than in the general population (37). 22.5 NATURAL HISTORY OF HIVASSOCIATED NAFLD The key histopathological event in the natural history of NAFLD is the development of liver fbrosis, that is, the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. This eventually leads to progressive distortion of the hepatic architecture and evolution to cirrhosis. The staging of liver fbrosis is essential for risk stratifcation and prognostication. Presence of stage 2 or higher liver fbrosis is an independent predictor of liver-related complications and all-cause mortality (69). Few studies investigated the natural history of NAFLD in the setting of HIV infection, and all of them are based on noninvasive diagnostic modalities rather than liver histology. The incidence rate of NAFLD reported in the general population varies from ranging from 2.8 to 5.2 per 100 PY (70). In HIV mono-infected patients, a few studies on the incidence of NAFLD have been performed so far. The incidence rate has been reported between 0 and 6.9 per 100 PY (95% CI, 5.9–7.9) (71,72). As for progression of liver fbrosis over time, the incidence of signifcant liver fbrosis was reported at 0.9 per 100 PY by the serum biomarker FIB-4, while the transition rate to signifcant liver fbrosis or liver cirrhosis by TE was determined at 12.7 per 100 PY (67,71). These fgures are higher than the general, uninfected population. Scarce data exist on clinical outcomes related to NAFLD in HIV mono-infected patients. In the general HIV-uninfected population, the 10-year mortality reported in 3,869 NAFLD subjects was 10.2%, higher than controls (7.6%) (73). A study of 1,092 patients from the LIVEr disease in HIV (LIVEHIV) Cohort reported an incidence rate of liver-related events of 8.6 per 1,000 PY, without difference between HIV mono-infected and HIV/HCV coinfected patients (74). Due to its systemic pathogenesis, NAFLD is a risk factor for all-cause mortality attributable to cardiovascular disease and extrahepatic cancer, as well as for incident T2DM, chronic kidney disease, vasculopathy (69,75–77). HIV itself and ART carry higher risk of these conditions (78). A study of 485 patients from the LIVEHIV

Cohort followed for a median of 40.1 months reported an increased incidence of T2DM and dyslipidemia in HIV mono-infected patients with NAFLD compared to those without NAFLD (79). The interplay between two multisystem diseases, HIV and NAFLD, could be responsible for these fndings. 22.6 DIAGNOSTIC TOOLS FOR NAFLD, LIVER FIBROSIS AND ESOPHAGEAL VARICES IN HIV MONO-INFECTED PATIENTS Several noninvasive tools for the diagnosis of hepatic steatosis and fbrosis have been extensively studies in NAFLD. These methods rely on two different approaches: a biological approach, based on the quantifcation of biomarkers in the serum, and a physical approach, based on the measurement of liver stiffness by either ultrasonographic or magnetic resonance elastography (80). Few of these methods have been validated against liver histology in the specifc setting of HIV infection. In 66 HIV monoinfected patients, Morse et al. found that TE outperformed the simple serum biomarkers APRI, FIB-4 and NAFLD fbrosis score to diagnose signifcant liver fbrosis. The reported area under the curve (AUC) for TE was 0.93, with associated sensitivity and specifcity of 93 and 73%, respectively, for a cutoff value of 7.1 kPa (81). Lemoine and colleagues reported on the diagnostic accuracy of several noninvasive diagnostic tests for hepatic steatosis, NASH and fbrosis in 49 HIV mono-infected patients with available histology (82). For the diagnosis of hepatic steatosis, the AUC for magnetic resonance imaging derived proton density fat fraction (MRI-PDFF) and CAP was 0.98 and 0.87, respectively. Interestingly, an ALT cutoff value of 36 had an AUC of 0.88 to diagnose NASH, with 91 and 77% sensitivity and specifcity, respectively. In this study the serum fbrosis biomarkers APRI and FIB-4 had higher performance than TE for the diagnosis of signifcant liver fbrosis. A poor concordance between serum fbrosis biomarkers and TE in the specifc setting of HIV infection has also been reported (83). CAP was also validated against MRI-PDFF in a study by Ajmera et al. including 70 HIV mono-infected patients, reporting an AUC of 0.82 and identifying an optimal cutoff value of 285 dB/m for CAP to detect at least 5% hepatic steatosis (84). The Baveno VII guidelines were proposed to reduce the number of unnecessary endoscopies: patients with compensated advanced chronic liver disease can forego esophagogastroduodenoscopy if the TE value is 150,000/μL (85). In a multicenter study, we validated these criteria in 507 PWH with TE >10 kPa, including 42 HIV mono-infected patients with suspected NAFLD. The Baveno VII criteria could save at least 33.3% screening endoscopy (86). These fndings can be used for resource optimization in HIV clinics. 22.7 MODELS OF CARE FOR HIVASSOCIATED NAFLD NAFLD is often asymptomatic until patients develop hepatic decompensation, with signifcant morbidity and mortality and related socioeconomic burden (8). There is a need for personalized medicine and implementation of strategies to identify those who have advanced liver fbrosis and who are at risk of adverse outcomes. The guidelines from the European AIDS Clinical Society (EACS) recommend the case fnding of advanced liver fbrosis in PWH with metabolic conditions or persistent elevated transaminases (87). These recommendations are 209

SECTION V: NAFLD IN SPECIAL POPULATIONS

in line with other at-risk populations for NAFLD-related liver fbrosis, such as patients with T2DM (88). In PWH, diagnosing liver fbrosis is challenging due to the large population at risk for NAFLD, as well as the additional resources of delivering TE with CAP, which is often not readily accessible in clinics practicing HIV care. Clinical pathways have been proposed in HIV-negative NAFLD to screen for liver fbrosis in at-risk populations and reduce the need for specialist tests. In these models, readily available and inexpensive fbrosis biomarkers, such as FIB-4, with high negative predictive value are used as frst-tier tests, while more specialized tests, such as TE with CAP, are second-tier tests reserved to cases in which fbrosis cannot be excluded by the simple biomarker (87). A recent international study in PWH applied several models based on serum fbrosis biomarkers as frst-tier tests, followed by TE with CAP. This care pathway would result in up to 86.3% reduction in need for TE examination, with increased accessibility and reduced costs (89). 22.8 TREATMENT OF HIV-ASSOCIATED NAFLD Lifestyle modifcations are the cornerstone of treatment for NAFLD, while limited pharmacologic options are available for patients with signifcant liver fbrosis. 22.8.1 Lifestyle Changes The frst line treatment for NASH is weight loss, through a combination of lifestyle changes including calorie reductions, exercise and healthy eating. In the general NAFLD population, suggested interventions for weight loss include 500–1,000 kcal energy defect to induce a weight loss of 500– 1,000 g/week (88). Weight loss of >7% can lead to resolution of NASH, while a weight loss >10% can regress liver fbrosis (88). Few ad hoc studies investigated the effect of lifestyle interventions on HIV-associated NAFLD. However, considering that BMI is a frequently reported predictor of NAFLD in PWH, these interventions may also be effective in PWH (87). A randomized controlled trial of PWH with NAFLD investigated telemedicine as a tool for dietary intervention during the COVID-19 pandemic. Fifty-fve PWH with NAFLD were allocated to dietary intervention vs. standard of care. During lockdown, 93.3% of patients in the standard of care group referred that the “diet got worse” vs. 6.7% in the intervention group, and 35.3% vs. 15.7% reported increase in appetite, respectively. PWH in the standard of care group gained more weight than in the intervention group (90). Another study evaluated the relationship between food intake of lipids with NAFLD in PWH. Participants with higher intake of total fat were associated with higher odds for NAFLD compared to those with lower consumption (adjusted odds ratio = 1.91, 95% CI 1.06–3.44) (91). Other important components of dietary interventions in the context of NAFLD include reducing alcohol intake, avoiding fructose-containing beverages and food, and limiting the consumption of processed red meat (88). 22.8.2 ART-Related Interventions The EACS guidelines recommend considering the use of metabolically neutral ART regimens in PWH at risk for or with NAFLD. In a randomized controlled trial, Macias and colleagues investigated the effect of switching efavirenz to raltegravir on hepatic steatosis diagnosed by TE with CAP among 39 HIV/HCV coinfected patients. At week 48, resolution of hepatic steatosis was observed in 47% patients who were switched to raltegravir compared to only 15% of

210

patients who were maintained on efavirenz (92). Similar results were obtained by an Italian observational study of 61 PWH, of whom those switched from ritonavir-boosted PIs to raltegravir had a signifcant decrease in hepatic steatosis (93). However, recent data suggest that regimens containing integrase inhibitors, in particular dolutegravir, may be associated with weight gain (45). Further studies with larger sample sizes and longer follow-up are warranted. In a retrospective cohort study, maraviroc, a chemokine receptor 5 antagonist, showed a potential protective role in reducing the incidence of NAFLD in PWH (94). 22.8.3 Pharmacologic Therapy Few pharmacologic approaches have been tested in the specifc context of HIV infection as PWH are currently excluded from global NASH clinical trials (39). In the context of HIV-associated lipodystrophy, pioglitazone reduced liver fat and lobular infammation in 13 PWH but did not achieve the primary endpoint of improvement or resolution of NASH (95). The ARRIVE trial, a double-blind, randomized, placebo-controlled trial, tested the effcacy of 12 weeks of treatment with Aramchol vs. placebo in 25 PWH with NAFLD. Over a 12-week period, there was no signifcant change of hepatic fat or body fat assessed by MRI-PDFF (96). In a phase 4 open-label clinical trial, vitamin E 800 IU daily for 24 weeks reduced ALT (−27 units/L), steatosis estimated by CAP (−22 dB/m), and cytokeratin 18 (−123 units/L) (24). A randomized multicenter trial including 61 PWH with NAFLD assessed the therapeutic potential of tesamorelin, a synthetic form of growth hormone-releasing hormone approved for the treatment of excess abdominal fat in HIV-associated lipodystrophy (97). After 12 months of treatment, steatosis had decreased by 32% from baseline in patients on the treatment arm, while it had increased by 5% in placebo patients. Moreover, 35% of patients in the tesamorelin group resolved steatosis in comparison to only 4% of patients on placebo. 22.8.4 Bariatric Surgery Bariatric surgery is an option for durable weight loss in obese NAFLD patients, with signifcant improvement in both associated metabolic syndrome comorbidities and liver fbrosis. Weight loss surgery is being increasingly considered in morbidly obese PWH (98). However, consideration should be provided to variable ART absorption after weight loss surgery (99). 22.9 CONCLUSION/SUMMARY POINTS NAFLD is frequent and more severe in the setting of HIV infection. A complex multifactorial pathogenesis underlines this epidemiologic evidence. Progression to NASH should be highly suspected in case of elevated ALT, overweight and confrmation by serum fbrosis biomarkers or TE. A case fnding of NAFLD-related liver fbrosis should be implemented at in PWH with metabolic comorbidities or persistently elevated ALT. A therapeutic approach in PWH should be based on lifestyle modifcation and a few pharmacologic options in the presence of NASH or signifcant liver fbrosis. Future research directions should target longitudinal studies characterizing the natural history of NASH and fbrosis progression, the identifcation of biomarkers to diagnose NASH in the specifc setting of HIV, along with targeted interventions to improve liverrelated clinical outcomes in PWH.

22 NAFLD IN HIV PATIENTS

22.10 CONFLICT OF INTEREST GS has acted as speaker for Merck, Gilead, Abbvie, Novonordisk, Novartis, served as an advisory board member for Merck, Novonordisk, Novartis, Gilead, Allergan and Intercept and has received unrestricted research funding from Theratec. 22.11 ACKNOWLEDGMENT GS is supported by a Senior Salary Award from FRQS (#296306). REFERENCES 1. Organization WH. www.who.int/gho/hiv/en/ Last access 9th May 2020, 2020. 2. CDC. Monitoring selected national HIV prevention and care objectives by using HIV surveillance data—United States and 6 dependent areas—2015. HIV Surveillance Suppl. Rep. 2017;22(2). 3. Lewden C, May T, Rosenthal E, Burty C, Bonnet F, Costagliola D, et al. Changes in causes of death among adults infected by HIV between 2000 and 2005: The “Mortalite 2000 and 2005” surveys (ANRS EN19 and Mortavic). J Acquir Immune Defc Syndr. 2008;48(5):590–598. 4. Smith CJ, Ryom L, Weber R, Morlat P, Pradier C, Reiss P, et al. Trends in underlying causes of death in people with HIV from 1999 to 2011 (D:A:D): A multicohort collaboration. Lancet. 2014;384(9939):241–248. 5. Rosenthal E, Salmon-Ceron D, Lewden C, Bouteloup V, Pialoux G, Bonnet F, et al. Liver-related deaths in HIV-infected patients between 1995 and 2005 in the French GERMIVIC joint study group network (Mortavic 2005 study in collaboration with the mortalite 2005 survey, ANRS EN19). HIV Med. 2009;10(5):282–289. 6. Croxford S, Kitching A, Desai S, Kall M, Edelstein M, Skingsley A, et al. Mortality and causes of death in people diagnosed with HIV in the era of highly active antiretroviral therapy compared with the general population: An analysis of a national observational cohort. Lancet Pub Health. 2017;2(1):e35–e46. 7. Kaspar MB, Sterling RK. Mechanisms of liver disease in patients infected with HIV. BMJ Open Gastroenterol. 2017;4(1):e000166. 8. Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, et al. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the study of liver diseases. Hepatology. 2018;67(1):328–357. 9. Eslam M, Sanyal AJ, George J, International Consensus P. MAFLD: A consensus-driven proposed nomenclature for metabolic associated fatty liver disease. Gastroenterology. 2020;158(7):1999–2014 e1. 10. Cervo A, Milic J, Mazzola G, Schepis F, Petta S, Krahn T, et al. Prevalence, predictors and severity of lean nonalcoholic fatty liver disease in HIV-infected patients. Clin Infect Dis. 2020. 11. Lake JE, Overton T, Naggie S, Sulkowski M, Loomba R, Kleiner DE, et al. Expert panel review on nonalcoholic fatty liver disease in persons with human immunodefciency virus. Clin Gastroenterol Hepatol. 2020. 12. Liu D, Shen Y, Zhang R, Xun J, Wang J, Liu L, et al. Prevalence and risk factors of metabolic associated fatty liver disease among people living with HIV in China. J Gastroenterol Hepatol. 2021;36(6):1670–1678.

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23 NAFLD in Liver Transplant Recipients Liyun Yuan and Norah Terrault CONTENTS 23.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 23.2 Graft and Patient Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 23.3 Prevalence of Metabolic Risks after Liver Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 23.3.1 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 23.3.2 Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 23.3.3 Hyperlipidemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 23.4 Immunosuppression in NAFLD and Post-Transplant Metabolic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 23.4.1 Corticosteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 23.4.2 Calcineurin Inhibitors (CNIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 23.4.3 Mammalian Target of Rapamycin (mTOR) Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 23.5 Recurrent or De Novo NAFLD: Incidence and Natural History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 23.6 Risk Factors for Development of Allograft NAFLD/NASH (Figure 23.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 23.6.1 Donor Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 23.6.2 Recipient Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 23.7 Monitoring for Recurrent or De Novo NAFLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 23.8 Management Approach for NAFLD Post-LT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 23.8.1 Immunosuppression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 23.8.2 Lifestyle Modifcations and Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 23.8.3 Role of Bariatric Surgery in Obese LT Recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 23.8.4 Repurposed Drugs for Treatment of Post-LT NASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 23.9 Management of Nonhepatic Risks among LT Recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 23.9.1 Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 23.9.2 Cardio- and Cerebrovascular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 23.9.3 Nonliver Malignancies Post-LT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 23.10 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Key Points 1. In the United States, the proportion of liver transplants (LT) performed for nonalcoholic steatohepatitis (NASH) more than quadrupled in the past 2 decades. 2. The post-LT survival of patients with a pre-LT diagnosis of NASH is similar or better than other indications such as alcohol and hepatitis C. Recurrent steatosis is common, but recurrent NASH cirrhosis is infrequent with follow-up durations of up to 10 years. 3. Post-transplant metabolic syndrome is very common, refecting comorbidities present in NASH patients preLT and the heightened risk of metabolic complications post-LT due to effects of immunosuppression and weight gain. 4. Monitoring for recurrent NAFLD and NASH is best accomplished using elastography with liver biopsy used to distinguish recurrent NAFLD/NASH from other post-LT liver complications, such as rejection and biliary disease. 5. As in nontransplant patients, the cornerstone of prevention of recurrent and de novo NAFLD is optimization of weight via lifestyle measures and treatment of metabolic comorbidities. Bariatric surgery in highly selected LT patients has yielded positive outcomes, but more data are needed. 6. Cardio- and cerebrovascular diseases and nonhepatic malignancies are the most frequent causes of death post-LT, so attention to surveillance and risk mitigation strategies is important. DOI: 10.1201/9781003386698-28

23.1 INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases globally (1), and NAFLD-associated cirrhosis is becoming a leading indication for liver transplantation (LT). In the United States, the proportion of liver transplant performed for nonalcoholic steatohepatitis (NASH) more than quadrupled between 2002 and 2019 from 5 to 28%. The rise in LT for NASH-associated hepatocellular carcinoma is noteworthy also, now the second most frequent cause of HCC among LT recipients in the US and the leading etiology of HCC among women undergoing LT (2). As the number of transplant recipients with NASH rises, attention has turned to understanding the unique risks of this population, who are older (3,4) at the time of LT and have higher rates of metabolic comorbidities such as obesity, diabetes and hypertension. These comorbidities add complexity to transplant management and heighten the risk of postLT complications such as cardiovascular disease, renal disease and malignancy (5). Additionally, post-transplant NAFLD is well-recognized, both recurrent (6) and de novo (7), with endogenous and exogenous factors contributing to its development and progression. Identifcation of potentially modifable risks and therapeutic strategies are important to improving post-transplant outcomes. 23.2 GRAFT AND PATIENT SURVIVAL The post-LT survival of patients with a pre-LT diagnosis of NASH is similar or better than other indications such as alcohol and hepatitis C. (8). In a recent analysis of the Scientifc Registry of Transplant Recipients of 6,515 LT 215

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for NASH cirrhosis between 2002 and 2019, the overall 5-year survival was 79% (9). In multivariable analysis, the independent predictors of lower survival were pre-LT diabetes, ventilator dependence, hemodialysis within a week of transplant, poor functional status and age older than 70; patients with all 5 of these factors had a 5-year survival less than 65%. Another study (10) using registry data found higher mortality among NASH vs. alcohol and hepatitis C etiologies within the frst year post-LT, with NASH patients having higher rates of death from cardiovascular and cerebrovascular diseases and age being a strong predictor of mortality: HR 1.31 (95% CI 1.04, 1.66) for patients 50–59 years, 1.66 (95% CI 1.31, 2.11) for patients 60–64 years, 2.08 (95% CI 1.63, 2.64) for patients 65–69 years, and 2.66 ((5% CI 1.98, 3.57) for patients and ≥70 years. Similarly, the US NailNASH consortium emphasized the importance of nonhepatic causes of death among patients transplanted for NASH. Among 938 patients followed for a median of 3.8 years post-LT and with 95 deaths, 19% were ascribed to infection, 18% to cardiovascular disease and 17% to cancer, with only 11% liver-related (11). Only 2.6% of deaths were attributable to recurrent NASH cirrhosis. In multivariable analysis, older age, end-stage renal disease and Black race were associated with inferior outcomes, and statin use with reduced risk of death (11). These studies highlight the importance of nonhepatic complications in patients transplanted for NASH and the need to consider means to modulate risk via patient selection and management to metabolic comorbidities. 23.3 PREVALENCE OF METABOLIC RISKS AFTER LIVER TRANSPLANTATION Metabolic syndrome is common post-LT, with prevalence increasing with time from LT (12). A recent meta-analysis, including 3,539 patients, reported a post-LT prevalence of metabolic syndrome of 39%, with pretransplant diabetes and obesity the only risk factors associated with its presence post-LT (13). However, it should be noted that this meta-analysis was focused on studies published prior to 2016 when NASH contributed only 10% or less to the study populations and were not representative of current LT cohorts, where NASH typically accounts for 25–30% of the LT recipients (2). Patients with NASH undergoing LT will have a higher likelihood of having obesity, diabetes, hypertension and dyslipidemia than patients with other etiologies (4). Thus it is not surprising that metabolic syndrome is exceedingly common among NASH transplant recipients— with a prevalence at 5 years post-LT of 90% (14). 23.3.1 Diabetes The vast majority of NASH patients with pre-LT DM will have persistent post-LT. A large retrospective study from Canada found NASH patients without diabetes pre-LT had a 2-fold higher likelihood of developing post-LT DM than non-NASH etiologies (15). In a national US cohort study of LT recipients without pre-LT DM, more with NASH vs. nonNASH (40 vs. 27%) had any documentation of de novo DM post-LT, with two-thirds within the frst 6 months (p < 0.0001) (16). Overall, NASH patients had a 30% higher risk of de novo DM than non-NASH, non-HCV controls, after accounting for other recognized risks including older age, use of calcineurin inhibitors, and body weight (aHR: 1.29, 95% CI: 1.18–1.42) (16). 23.3.2 Obesity Patients on the waiting list suffer from poor appetite, micronutrient defciencies and protein-calorie 216

malnutrition, refecting the hypercatabolic state of advanced cirrhosis. However, post-LT patients rapidly regain their appetite and rediscover the joys of eating after months to years of early satiety due to ascites, dietary sodium restrictions and other factors, resulting in the risk of excess weight gain (17). In the frst 1–3 years after LT, average weight gain is 5–10 kg (18–20). Interestingly, the surgery itself may be a factor, with transection of the autonomic nerves of the liver, both the anterior plexus around the hepatic artery and the posterior plexus around the portal vein and bile duct (21). Potential effects on appetite and homeostasis are suggested by studies comparing LT and kidney transplant recipients, with LT patients having higher fat but lower carbohydrate intake, hyperphagia and loss of increased thermogenesis despite increasing body mass, all of which contribute to excessive weight gain (22). Additionally, the majority of patients do not resume or achieve normal levels of physical activity, thus compounding the risk for excessive weight gain post-LT. In a cross-sectional study of 156 patients more than a year from LT, 63% had metabolic syndrome, and there was an inverse correlation between presence of metabolic syndrome and level of physical activity (aOR = 0.69, 95% 0.54–0.89) (23). A small study comparing fuel utilization in NASH vs. non-NASH LT recipients showed that NASH patients have impaired fatty acid utilization that was correlated with fat free muscle volume and visceral adiposity (24). The authors proposed that this was a marker of reduced metabolic fexibility that may predispose to development of recurrent NAFLD. Whether patients with NASH are at greater risk for weight gain postLT than patients with other etiologies has not been rigorously evaluated. Two single-center studies from Europe found no difference in overweight/obesity at 5 years post-LT in NASH compared to alcohol-associated liver disease (19, 25). 23.3.3 Hyperlipidemia The prevalence of hyperlipidemia is estimated to vary from 27 to 71% among LT recipients (26). Atherogenic dyslipidemia, defned as elevated small dense LDL-C and the increased size and concentration of VLDL particles, is very common among LT recipients with NAFLD or metabolic syndrome with reported prevalence of up to 80% (6,26,27). A prospective study of 130 enrolled LT recipients (28) showed serum small dense LDL-C >25 mg/ dl was a strong predictor for post-LT cardiovascular events (hazard ratio 6.4, 95% CI 2.7, 15.3; P < 0.001), while LDL-C cutoff of 100 mg/dl was unable to identify of CVD risk among this group. The development of post-LT atherogenic dyslipidemia is infuenced by post-LT weight gain, insulin resistance, de novo NAFLD, abnormal renal function and genetic predisposition in a couple of the impact of immunosuppression (29,30), specifcally cyclosporin, which affects lipid metabolisms by increasing triglyceride secretion, reducing bile acid synthesis and reducing lipolysis (28,31). 23.4 IMMUNOSUPPRESSION IN NAFLD AND POSTTRANSPLANT METABOLIC SYNDROME The need for chronic immunosuppression for prevention of allograft rejection and insuring long-term graft survival places patients at risk for metabolic complications. Most of the drug classes commonly used for immunosuppression have some metabolic consequences (Table 23.1), and for patients with preexisting metabolic comorbidities, immunosuppressive drugs can exacerbate risk of metabolic complications post-LT.

23 NAFLD IN LIVER TRANSPLANT RECIPIENTS

Table 23.1: Metabolic Side Effects of Immunosuppressive Drugs Immunosuppressions Corticosteroids

Calcineurin inhibitors (CNIs)

Mammalian target of rapamycin (mTOR) inhibitor

Actions Hyperphagia ↑Appetite ↑De novo lipogenesis ↑Glucogenesis ↑Insulin resistance ↑Vascular resistance ↓β cell function ↓Peripheral glucose utilization ↓Mitochondrial β oxidation ↓Triglyceride secretion ↓Bile acid synthesis ↑Renal arterial vasocontriction ↓GFR and ↑sodium retention ↓β cell mass ↓Hepatic insulin clearance ↑Hepatic gluconeogenesis ↑Hepatic synthesis of triglyceride ↑Secretion of VLDL ↑FFA release from adipose

23.4.1 Corticosteroids Corticosteroids are commonly used in the early postLT period to treat episodes of acute rejection. Excess exogenous or endogenous glucocorticoid has been implicated across all the stages of NAFLD pathogenesis. Glucocorticoid binds to glucocorticoid receptors, acting upon both liver and adipose tissues by regulating lipid metabolism (32). In adipose tissue, it promotes lipolysis, increases the delivery of free fatty acids for de novo lipogenesis and drives peripheral insulin resistance. Within the liver, glucocorticoid promotes gluconeogenesis and glycogenolysis, increasing the availability of glucose as a substrate for de novo lipogenesis. Steroid avoidance or minimization is viewed as a favorable strategy to prevent excessive weight gain, though data supporting such a beneft are mixed (33–35). 23.4.2 Calcineurin Inhibitors (CNIs) CNIs are the backbone of immunosuppression for LT recipients but can exacerbate multiple facets of posttransplant metabolic syndrome. In the US, tacrolimus is the most widely prescribed CNI, with cyclosporine use very infrequently. Diabetes is more frequent with tacrolimus than cyclosporine (36), whereas hypertension and renal dysfunction are more common with cyclosporine than tacrolimus (37). CNIs promote insulin resistance by impairing beta cell function, decreasing insulin secretion and enhancing insulin sensitivities. They affect lipid metabolism by impairing mitochondrial beta-oxidation and decreasing TG secretion (38). CNIs cause hypertension through arteriolar vasoconstriction, and calcium blockades are the most effective antihypertensive agent for CNIs-induced hypertension. Direct comparison of CNIs side effects in NASH vs. non-NASH post-LT recipients is lacking, but since NASH patients have a higher prevalence of metabolic syndrome pre-LT, an increased risk for CNI-related toxicities post-LT should be anticipated. Minimization of CNI dosage to reduce the long-term metabolic consequences is the goal in any LT patient but is particularly important for the NASH LT recipient.

Metabolic Consequences Weight gain DMII and HTN

Weight gain Insulin resistance/type 1 diabetes Renal dysfunction Hyperlipidemia

Hyperlipidemia Insulin resistance

23.4.3 Mammalian Target of Rapamycin (mTOR) Inhibitor The mTOR inhibitors, including sirolimus and everolimus, were initially introduced as renal-sparing immunosuppression. The early use of everolimus (started at 1 month post-transplant), in combination with reduced dose tacrolimus, has been shown to have a benefcial effect on renal function, particularly in those with reduced eGFR. Their antineoplastic effects are viewed as potentially benefcial to patients who have undergone LT for liver cancer (39, 40), with a recent metanalysis showing a survival beneft (41). As the proportion of patients with NASH undergoing LT with HCC as their primary indication increases, there may be reason to consider an immunosuppressive regimen that includes an MTOR inhibitor. Yet adverse effects are recognized, with dyslipidemia observed in over 50% patients (42). Thus, for NAFLD patients, the benefts of preserving renal function and potentially reducing HCC recurrence needs to be balanced with the worsening dyslipidemia. 23.5 RECURRENT OR DE NOVO NAFLD: Incidence and Natural History Both recurrent NAFLD and de novo NAFLD are recognized post-LT, with the distinction dependent upon whether or not there was a pre-LT diagnosis of NASH. A pretransplant diagnosis is typically based on the presence of risk factors and exclusion of other causes, with liver biopsy often not done. The loss of classic histologic features of NASH with advanced cirrhosis is recognized. In a detailed study of explants form 2014 to 2016, 3 of 20 patients (15%) with a pre-LT diagnosis of NASH lacked histologic features to confrm the diagnosis, and, on the fip side, of 37 patients labeled as cryptogenic cirrhosis, 7 (19%) had histologic features of NASH (43). Accurate pre-LT diagnosis may be relevant in predicting the risk of post-LT NASH. In a single-center study of 258 patients with a pretransplant diagnosis of cryptogenic cirrhosis or NASH who underwent protocol biopsies post-LT, recurrent steatosis was more frequent in the NASH (45%) than cryptogenic (23%) group at 5 years (44). This suggests that 217

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the evaluation of explants for evidence of NASH may help to predict the likelihood of recurrent disease. The histopathology features of allograft NASH are the same as the native liver, with immunosuppression per se not believed to alter the histologic presentation. However, concurrent allograft complications, such as chronic rejection or chronic biliary strictures, may infuence the risk for fbrosis progression. For patients with pretransplant NASH, allograft steatosis typically appears within the frst year (45) (Figure 23.1). This rapid development of steatosis within the frst year post-LT is not surprising, given that LT reverses the chronic catabolic state of advanced cirrhosis and there is increased appetite and associated weight gain starting soon after LT and that the doses of immunosuppressive drugs are higher in the early vs. late post-LT period. These factors set the stage for metabolic derangements that favor steatosis. In a meta-analysis of 17 retrospective studies and including 2,378 patients, the estimated incidence of recurrent NAFLD at 1, 3 and 5 years was 59, 57 and 82% and for recurrent NASH was 53, 57.4 and 38%, respectively, but with low confdence in these estimates due to study heterogeneity (46). In contrast, de novo recurrent NASH was infrequent with an estimated incidence of only 17% at 5 years. A higher frequency of advanced fbrosis (F3/F4) at 5 years has been reported in those with recurrent vs. de novo NASH (72% vs. 13%, p < 0.01) (45). Whether disease progression is accelerated post-LT in those who develop recurrent NASH is unclear. Though recurrent NASH is very common, development of allograft dysfunction or cirrhosis is very low (Figure 23.1). In a retrospective cohort of 226 patients, with 75% undergoing at least 1 biopsy during post-LT follow-up, 49% developed recurrence of NASH at an average 3 years, with 23% developing advanced fbrosis and 1.8% progressing to cirrhosis at a mean of 9 years follow-up (47). Refecting this low rate of cirrhosis at 10 years post-LT, studies of post-LT survival show no difference for those with NAFLD vs. other indications (48). While the risk of recurrent cirrhosis and its complications may be a threat to patient and graft

survival with longer duration of follow-up (beyond 10 years), competing risks of mortality among patients with NAFLD are likely to continue to be the primary drivers of post-LT mortality—specifcally cardio- and cerebrovascular diseases (11, 49). Moreover, as the proportion of NASH LT recipients with HCC as a primary indication for LT increases, outcomes will be strongly infuenced by rates of recurrent HCC (50, 51). 23.6 RISK FACTORS FOR DEVELOPMENT OF ALLOGRAFT NAFLD/NASH (FIGURE 23.1) 23.6.1 Donor Factors The prevalence of NAFLD among donors is increasing, and up to 70% of utilized donors have some degree of steatosis. Whether the presence of donor steatosis is a risk factor for de novo or recurrent NAFLD is unclear. In a biopsybased study from France, with 31% of 599 patients having steatosis (53% grade 1; 16% grade 3), the presence of donor steatosis was an independent risk factor for post-NAFLD (52). Similarly, in a study of 155 Korean LT recipients with a liver biopsy beyond 1 year, donor steatosis was associated with a 3-fold higher odds of recipient NAFLD (OR 3.15, p = 0.02) (53). However, a US study conducted with paired donor–recipient liver biopsies (only 14% with pre-LT NASH) found no association between donor steatosis and subsequent NAFLD with follow-up an average 704 days post-LT (54). Donor genetic polymorphisms increase the likelihood of liver allograft fat content after LT (55). In a single-center study from the Czech Republic (56,57) consisting of 268 adult LT donors/recipients with genotyping and at least 1 liver biopsy taken 6–30 months after LT, the carrier state for the donor TM6SF2 A and the PNPLA3 G alleles, recipient age, pretransplant BMI, and presence of DM were strong predictors of increased liver graft steatosis after LT. The frequency of allograft steatosis was 36.7, 53.2, 58.3, and 77.8% in those without risk alleles, with donor PNPLA3 G, with donor TM6SF2 A, and both risk alleles, respectively (56,57). In another study where the prevalence of donor

Figure 23.1 Donor, recipient and post-transplant factors related to immunosuppressive drugs infuence the natural history of NAFLD after liver transplant. Approximately 20-25% will develop recurrent NASH within 5 years, with rates of cirrhosis infrequent in frst 10 years post-transplant. 218

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PNPLA3 G allele was low, recipients homozygous for G alleles had a 13.7-fold higher risk of graft steatosis than recipients with normal PNPLA3 CC genotype (P = 0.022), independent of recipient age, weight gain after liver transplantation, or the underlying disease (58). Thus the future risk predictor for the LT recipient may need to consider donor and recipient genetic polymorphisms. 23.6.2 Recipient Factors As in the nontransplant setting, the metabolic syndrome is associated with recurrent and de novo steatosis. The majority of NASH LT recipients have preexisting obesity, diabetes, dyslipidemia and history of hypertension. These persist and are often exacerbated post-LT. Rapid weight gain after liver transplantation is an important contributing factor to de novo NAFLD or recurrent NAFLD. Richards et al. (59) shows the greatest weight gain occurred within 6 months after transplant. Post-transplant weight gain is associated with increased risk of hypertension, hypercholesterolemia, hypertriglyceridemia and insulin resistance. In a meta-regression of factors associated with post-LT NASH, post-LT body mass index, dyslipidemia, and history of alcohol use were identifed (26). Whether specifc metabolic risk increases the likelihood of NASH (vs. steatosis only) is unclear, as the overall proportion of patients developing NASH is modest, and there are limited longitudinal biopsy studies upon which to evaluate the relative contributions of weight gain, diabetes, dyslipidemia and hypertension over time—especially as patients may experience periods of exacerbation and control. Among nontransplant patients, disparities in natural history are evident by sex and race-ethnicity. Latinx have the highest prevalence of NAFLD, followed by Caucasians and Asian (60). Although African Americans have a high prevalence of metabolic syndrome, NAFLD prevalence is low. Women have lower rates of NASH progression until menopause, after which the gender gap closes (61). Whether these disparities infuence rates of recurrent or de novo NASH post-LT requires further investigation. As most NASH LT recipients are over the age 50, sex differences are unlikely, but race-ethnicity disparities warrant additional study. 23.7 MONITORING FOR RECURRENT OR DE NOVO NAFLD Liver biopsy remains the gold standard for diagnosis of NAFLD and quantifying stage of fbrosis in the post-LT setting. For patients with elevated liver tests, the need to exclude other potential allograft complications, including acute rejection or viral infections, requires the use of liver biopsy. However, beyond the early transplant period, risk for other allograft complications declines and monitoring for recurrent NASH is typically done using noninvasive methods. Noninvasive serum biomarkers, such as APRI and FIB4 are not accurate in the post-LT setting (62), primarily because platelet counts may be affected by immunosuppression and residual portal hypertension, and AST/ALT levels may be increased by other non-NAFLD post-transplant allograft complications, such as biliary strictures. The use of elastography offers more promise, though the presence of concurrent liver conditions may lead to erroneous measures. Liver stiffness may be affected by cholestasis (from chronic rejection), vascular compromise, biliary obstruction or anatomic variability. However, in patients without those allograft complications (the majority), elastography measures of steatosis and fbrosis may be useful. Published series suggest the liver stiffness cutoffs for signifcant or advanced fbrosis are different for liver

allograft compared to the native liver. In a single-center study (63) of 150 LT recipients evaluated using transient elastography (TE) with controlled attenuation parameter (CAP) a mean of 10 years post-LT and using CAP cutoff of 222 dB/m to defne steatosis, 70% of patients were affected, with 28% of patients meeting the criteria for severe steatosis (CAP of ≥290 dB), and most patients had normal liver enzymes. Advanced fbrosis was identifed in 12.7%, and of the 5 patients who underwent liver biopsy, none showed cirrhosis but all had evidence of chronic rejection. A systemic review suggests higher cutoffs of liver stiffness for advanced fbrosis in LT recipients, varying from 10.5 to 26.5 kPa (64), but much of the published studies focus on patients with hepatitis C. In a recent large prospective study of 259 LT recipients who had TE at the time of liver biopsy, the optimal cutoff values were suggested to be 15.1 kPa for F3 and 16.7 kPa for F4 for LT recipients for all etiologies (65). More studies are needed, but available data suggest that TE has value in identifying steatosis, and an elevated liver stiffness measure should prompt consideration of liver biopsy to evaluate for cause. The magnetic resonance elastography (MRE) and proton density fat fraction (PDFF) methods have high accuracy in detection of liver fbrosis and steatosis in the nontransplant setting (66,67). In a systemic review by Singh et al. including 6 cohorts and 141 LT recipients with MRE and liver biopsy, a mean AUROC value of MRE for advanced fbrosis and cirrhosis were 0.83 (95% CI: 0.61–0.88) and 0.96 (0.93–0.98), respectively, whereas the accuracy of mild to moderate fbrosis was lower, AUROC was 0.73 (0.66–0.81) and 0.69 (0.62–0.74) (68). However, almost all included patients had HCV or alcohol-associated liver diseases. A cross-sectional study of 126 LT patients (50% for HBV, only 9% for NAFLD) who underwent TE and MRE at a mean of 75 months post-LT, showed that MRI-PDFF and TE CAP were moderately correlated (r = 0.44) but higher for identifying signifcant fbrosis (r = 0.79) (69). Thus MRE/PDFF or TE/CAP can be considered for monitoring steatosis and fbrosis post-LT. Given that most patients have normal liver enzymes, reliance on liver test abnormalities to trigger evaluation will miss patients with signifcant disease. We proposed an algorithm of surveillance (Figure 23.2). We favor the use of TE/CAP due to its ease and lower cost than MRE/PDFF and suggest that programs consider a protocolized approach with imaging done at 1 year and then every 2–3 years depending on initial fndings. Patients with severe steatosis warrant closer follow-up, as do those with evidence of increased liver stiffness. Liver biopsy still has a role to play, particularly in distinguishing recurrent or de novo NASH from other post-LT complications. 23.8 MANAGEMENT APPROACH FOR NAFLD POST-LT Prevention of recurrent or de novo NAFLD depends frst and foremost on prevention and optimization of metabolic complications (Figure 23.2). Immunosuppression warrants careful attention, as most of the drugs used to prevent rejection have adverse metabolic side effects. Lifestyle measures that focus on attaining a more ideal body weight through healthy diet and exercise are important with approaches similar to that used in nontransplant settings, though one must acknowledge that studies on the effcacy of dietary and exercise measures in LT recipients are lacking. Bariatric surgery may be considered in the setting of LT. Repurposed drugs, such as semaglutide, may have a role. Finally, given the heightened 219

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Figure 23.2 Suggested approach to monitoring patients at risk for recurrent NAFLD focused on use of hepatic elastography. Prevention requires attention to lifestyle measures starting early after liver transplantation to avoid rapid weight gain, screening and treating metabolic comorbidities that are more frequent post-LT, and use of NAFLD-specifc treatments when recurrent NAFLD occurs. cardiovascular risk of LT recipients, particularly those with metabolic comorbidities, transplant physician plays an important role in advocating for use of medications to reduce risk, such as statins, and to assist in managing drug–drug interactions. 23.8.1 Immunosuppression While corticosteroids are often used at high doses in the early post-LT period and for treatment of acute rejection, long-term corticosteroids are to be avoided. As CNIs carry the greatest risk of metabolic complications and risk for renal dysfunction, strategies to minimize CNIs are desirable in this population. Reduction of CNIs has been shown to improve post-LT metabolic syndrome (70, 71). Reduced dose CNIs in combination with mTOR inhibitors or with mycophenolate are potential approaches. A randomized study of LT patients (72) was divided into 3 treatment groups: everolimus plus reduced tacrolimus (n = 245); tacrolimus control (n = 243); and tacrolimus elimination with everolimus alone (n = 231). The groups with tacrolimus reduction/elimination achieved less weight gain, better renal function and improved metabolic profles. Reduction of CNI by replacement with mycophenolate mofetil has been shown to improve posttransplant dyslipidemia, renal function and hypertension (70). 23.8.2 Lifestyle Modifcations and Exercise Lifestyle modifcation remains the mainstay and frst-line therapy for prevention and treatment of post-LT NAFLD. Establishing healthy eating patterns early post-LT is important, particularly for individuals with pre-LT NASH. Nutritional consultation in the early post-LT period may be benefcial. A well-balanced Mediterranean diet with whole grain, vegetable, fruits, fsh, beans and nuts diet and 220

elimination of fructose are among the key recommendations. Coffee is benefcial for many liver-related outcomes and drinking 3 or more cups per day may be benefcial (73)—though data specifc to LT patients are lacking. Exercise is pivotal for improving overall post-transplant health, as well as reduction in risk of metabolic syndrome. LT recipients often experience an initial downward trajectory in frailty in the immediate post-LT periods, typically the low point being at month 3, followed by a gradual modest improvement by 12 months post-LT (74). In a study of 204 LT recipients, exercise intensity was inversely associated with development of post-LT metabolic syndrome (OR = 0.69, 95% CI = 0.54–0.89). Three randomized trials of post-transplant exercises demonstrated that any forms of resistance exercise, such as home-based exercise or structured exercise programs or aerobic ftness, when adhered to for a long period of time, provides beneft in improving global muscle strength, pulmonary function and body composites (75–77). An exercise program needs to be individualized, as each LT recipient has a distinct course of recovery, infuenced by age, fragility, comorbidities and post-transplant complications. US population guidelines recommend 150 minutes/week of moderate-to-vigorous activity, along with resistance exercise training for 15–20 minutes twice a week (78), and this is a reasonable target for post-transplant recipients. A personized training program tailored to the need of the transplant recipients is ideal, but an emphasis on physical activities from care givers, providers and social groups is indispensable to success. 23.8.3 Role of Bariatric Surgery in Obese LT Recipients Data are limited to single-center experiences but highlight the potential metabolic benefts as well as weight improvements. The University of California–San Francisco has the

23 NAFLD IN LIVER TRANSPLANT RECIPIENTS

largest experience with pre-LT laparoscopic sleeve gastrectomy in highly select patients with stable cirrhosis and well-controlled complications (79,80). In long-term follow-up (mean 5 years) of patients with pre-LT sleeve gastrectomy who subsequently underwent LT, those who had bariatric surgery had a signifcantly lower risk post-LT of diabetes (OR 0.04, 95% CI 0.00–0.41, p = 0.01), hypertension (OR 0.15, 95% CI 0.04–0.67, p = 0.01), and recurrent and de novo NAFLD (HR 0.19, 95% CI 0.04–0.91, p = 0.04) that matched patients who were managed medically. The Mayo Clinic has the largest experience with simultaneous sleeve gastrectomy and LT (81). In a highly selective group of morbidly obese candidates, weight loss of 20 to 50% of initial body weight was achieved after 3 years of follow-up, and none of the patients had major complications. Finally, the largest singlecenter experience post-LT bariatric surgery comes from the University of Cincinnati and included 15 patients with median time from LT to laparoscopic sleeve gastrectomy of 2.2 years (82). They showed BMI decreased from 42.7 to 35.9 kg/m2 with a reduction of insulin doses among insulindependent diabetics with follow-up period of 2.6 years. While these early experiences are encouraging, it must be emphasized that this represents the results of highly experienced centers and in highly select patients only. In a meta-analysis (83) including a total of 96 patients from 8 studies who underwent bariatric surgery, 28 were prior to the transplant, 29 simultaneous with LT, and 39 after LT. The bariatric surgery-related morbidity and mortality rates were 37 and 0.6%, respectively. Importantly, improvement of hypertension and diabetes were seen in 61 and 45% of patients, respectively. Sleeve gastrectomy was the preferred technique in most studies and is favored because it preserves the anatomical continuity with the duodenum allowing for ease of biliary tree access and lowers the risk of malnutrition and malabsorption, and absorption of immunosuppressive medications is largely unaffected. Unresolved issues include the best timing of the procedure (pre-LT, concurrent with LT or post-LT), defning the criteria for optimal candidates and full characterization of short- and long-term risks. 23.8.4 Repurposed Drugs for Treatment of Post-LT NASH There are no published clinical trials of therapies for prevention or treatment of allograft NASH. However, data gleaned from the non-LT populations may be considered. The PIVENS clinical trial showed that both vitamin E 800 IU daily and pioglitazone are associated with improvement in histologic features of NASH, supporting a potential role in post-LT care (84). Neither drug interacts with calcineurin inhibitors. Cautions with vitamin E include avoidance in those with diabetes (less well-studied) and in those without risk for prostate cancer or stroke. Pioglitazone (85) is limited by the side effects of edema and weight gain, which many patients fnd diffcult to accept, given emphasis on weight optimization. Glucagonlike peptide-1 receptor agonists (GLP-1RAs), liraglutide and semaglutide are FDA-approved medical therapy for obesity or type II DM. In a 72-week, double-blinded phase 2 trial involving biopsy-confrmed NASH and fbrosis (86), 59% patients with 0.4 mg once-daily subcutaneous semaglutide achieved NASH resolution, compared to 17% in the placebo group. A dose-dependent weight reduction is seen with treatment with semaglutide. Seventy-two-week treatment with 0.4 mg daily dose achieves 13% body weight loss along with improvement of metabolic syndromes.

Weekly subcutaneous dose, 2.4 mg of semaglutide was also approved for obesity treatment, and approximately average 16% body weight reduction was observed with a 68-week course (87). Gastrointestinal adverse effects are very common, including nausea, abdominal pain and diarrhea. Although the clinical trials have not been extended to post-transplant recipients, owing to the therapeutic effcacy in weight loss, improvement of lipid panel, insulin resistance and type 2 DM, semaglutide may be a consideration in LT recipients with NASH. 23.9 MANAGEMENT OF NONHEPATIC RISKS AMONG LT RECIPIENTS Among NASH LT recipients, morbidity and mortality related to nonhepatic complications are noteworthy. In a multicenter US study of NASH recipients followed for a median of 3.8 years (IQR 1.6–7.1), the rank order of causes of death were infection, cardiovascular disease and cancer, with liver-related mortality ranking 4th and accounting for only 11% of deaths (11). In multivariable analysis, older age, end-stage renal disease and Black race were associated with inferior outcomes, and statin use reduced risk of death. Such studies highlight the importance of transplant physicians supporting a strategy of risk mitigation for cardiovascular, infectious and cancer complications in NASH recipients. 23.9.1 Infections The risks for heightened morbidity and mortality from infectious complications are likely multifactorial, including older age, greater frailty after LT (88), concurrent diabetes and higher rates of renal dysfunction. Prevention of frailty through dietary and physical activity interventions may be of particular importance to NASH patients. 23.9.2 Cardio- and Cerebrovascular Diseases In the NailNASH multicenter study, the most common cardiovascular complications seen post-LT were atrial fbrillation (11%), cardiac dysrhythmia (18%), ischemic heart disease (3%) and congestive heart failure (10%) (11). The authors highlight that, despite careful pretransplant cardiac evaluation and exclusion of those with signifcant atherosclerotic disease, events rates related to ischemic heart disease doubled (from 1.4 to 2.7%) over the study period of ~4 years follow-up. Moreover, statins were shown to have a positive infuence on post-LT outcomes. Statin therapy is the cornerstone for prevention of atherosclerotic cardiovascular disease. The development of dyslipidemia accelerates after LT with the prevalence increasing from 23% pretransplant to 55.5% at 5 years (89). Up to 70% patients on mTOR inhibitors will develop elevated triglycerides and low-density lipoprotein cholesterol. This highlights the importance of statin therapy after LT to optimize lipid panel and decrease risk of cardiovascular events. A potential additional beneft of statin therapy among LT recipients includes risk reduction of venous thromboembolic events and hepatic artery complication (90). However, less than half of statin-eligible LT recipients receive statin therapy, and doses of statins are often underprescribed (91). Statins are well-tolerated among LT recipients, but attention to potential drug–drug interactions is necessary. CNIs, particularly cyclosporin, have a dominant inhibitory effect on a liver-specifc statin transporter and increases the level of most statins as they share the common cytochrome P450 metabolism pathway. Pravastatin and rosuvastatin are less affected through the P450 systems and thus are favorable choice post-organ transplantation. 221

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Beyond this, there may be potential hepatic beneft of statin therapy across all the stages of NAFLD spectrum. Population-based studies in nontransplant patients show that statin treatment decreases the risk of NAFLD development and progression (92) and is associated with reduced risk of HCC among patients with NAFLD (93). Even among patients with cirrhosis, statins appear to be benefcial in improving portal hypertension, reducing risk of HCC and improving overall mortality (94,95). Collectively, these data serve to support the use of statins in post-LT patients, with indications similar to non-LT recipients. 23.9.3 Nonliver Malignancies Post-LT NASH LT recipients have a heightened risk of de novo malignancy, which has been underappreciated. Data from the SRTR found that patients transplanted for NASH had the highest rates of de novo malignancies post-LT at 16.3% (95% CI, 13.9–18.7) at 10 years and driven principally by the higher risk of skin and solid organ malignancies (96). Recipients who underwent transplant for NASH had the highest risk of breast cancer, colon cancer (among nonPSC etiologies) and pancreatic cancer. The NailNASH US cohort of 938 NASH patients transplanted 1997–2007, de novo malignancies accounted for 17% of deaths. As NASH LT recipients are older, on average, than non-NASH recipients, the increased rate of nonliver malignancies, in part, refects age-related risk. However, as nonliver malignancies occur at higher rates among non-LT NASH patients than among non-NASH, there is likely a true increase in risk of nonliver malignancies among NASH LT recipients, and vigilance for de novo malignancies is warranted (96). 23.10 SUMMARY NASH-associated cirrhosis and HCC are increasingly prevalent among LT candidates. As a patient population, these patients are older and more frequently have metabolic comorbidities that can contribute to post-LT morbidity and mortality. Recurrent NAFLD is common, though progression to NASH and NASH-associated cirrhosis is slow, with few developing liver disease complications within the frst decade post-LT. Cardio- and cerebrovascular complications and nonliver malignancies are more frequent causes of death post-LT, highlighting the need for post-LT surveillance and prevention strategies to maximize long-term survival. Interventions are modeled after the practices in non-LT patients with NASH, with focus on weight optimization through diet and exercise and on aggressive management of hypertension, dyslipidemia and diabetes. Attention to immunosuppression is key. Bariatric surgery can be a consideration in select patients. NASH-specifc therapies are understudied in LT recipients, and consideration of this special population for future clinical trials is important—with the goal of preventing NASH progression and insuring long-term graft survival. REFERENCES 1. Z. Younossi et al., Global burden of NAFLD and NASH: Trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 15, 11–20 (2018). 2. Z. M. Younossi et al., Nonalcoholic steatohepatitis is the most rapidly increasing indication for liver transplantation in the United States. Clin Gastroenterol Hepatol 19, 580–589.e585 (2021). 3. J. B. Henson et al., Transplant outcomes in older patients with nonalcoholic steatohepatitis compared 222

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61. J. D. Yang et al., Gender and menopause impact severity of fbrosis among patients with nonalcoholic steatohepatitis. Hepatology 59, 1406–1414 (2014). 62. I. Mikolasevic et al., Noninvasive markers of liver steatosis and fbrosis after liver transplantation—Where do we stand? World J Transplant 11, 37–53 (2021). 63. M. Chayanupatkul, D. B. Dasani, K. Sogaard, T. D. Schiano, The utility of assessing liver allograft fbrosis and steatosis post-liver transplantation using transient elastography with controlled attenuation parameter. Transplant Proc 53, 159–165 (2021). 64. K. Kohda, N. Sawada, Y. Kawazoe, Formation of O6,7-dimethylguanine residues in calf thymus deoxyribonucleic acid treated with carcinogenic N-methylN-nitrosourea in vitro. Chem Pharm Bull (Tokyo) 39, 801–802 (1991). 65. B. Della-Guardia et al., Diagnostic accuracy of transient elastography for detecting liver fbrosis after liver trannsplantation: A specifc cut-off value is really needed? Dig Dis Sci 62, 264–272 (2017). 66. C. C. Park et al., Magnetic resonance elastography vs transient elastography in detection of fbrosis and noninvasive measurement of steatosis in patients with biopsy-proven nonalcoholic fatty liver disease. Gastroenterology 152, 598–607.e592 (2017). 67. B. Taouli, L. Serfaty, Magnetic resonance imaging/ elastography is superior to transient elastography for detection of liver fbrosis and fat in nonalcoholic fatty liver disease. Gastroenterology 150, 553–556 (2016). 68. S. Singh et al., Diagnostic accuracy of magnetic resonance elastography in liver transplant recipients: A pooled analysis. Ann Hepatol 15, 363–376 (2016). 69. Z. Melekoglu Ellik et al., Evaluation of magnetic resonance elastography and transient elastography for liver fbrosis and steatosis assessments in the liver transplant setting. Turk J Gastroenterol 33, 153–160 (2022). 70. D. D’Avola et al., Cardiovascular morbidity and mortality after liver transplantation: The protective role of mycophenolate mofetil. Liver Transpl 23, 498–509 (2017). 71. K. D. Watt, Metabolic syndrome: Is immunosuppression to blame? Liver Transpl 17(Suppl 3), S38–S42 (2011). 72. P. De Simone et al., Everolimus with reduced tacrolimus improves renal function in de novo liver transplant recipients: A randomized controlled trial. Am J Transplant 12, 3008–3020 (2012). 73. J. W. Molloy et al., Association of coffee and caffeine consumption with fatty liver disease, nonalcoholic steatohepatitis, and degree of hepatic fbrosis. Hepatology 55, 429–436 (2012). 74. J. C. Lai et al., Physical frailty after liver transplantation. Am J Transplant 18, 1986–1994 (2018). 75. A. M. Garcia, C. E. Veneroso, D. D. Soares, A. S. Lima, M. I. Correia, Effect of a physical exercise program on the functional capacity of liver transplant patients. Transplant Proc 46, 1807–1808 (2014). 76. D. Moya-Nájera et al., Combined resistance and endurance training at a moderate-to-high intensity improves physical condition and quality of life in liver transplant patients. Liver Transpl 23, 1273–1281 (2017). 77. J. C. Jones, J. S. Coombes, G. A. Macdonald, Exercise capacity and muscle strength in patients with cirrhosis. Liver Transpl 18, 146–151 (2012). 78. C. J. Lavie et al., Exercise and the cardiovascular system: Clinical science and cardiovascular outcomes. Circ Res 117, 207–219 (2015). 224

79. S. R. Sharpton, N. A. Terrault, M. M. Tavakol, A. M. Posselt, Sleeve gastrectomy prior to liver transplantation is superior to medical weight loss in reducing posttransplant metabolic complications. Am J Transplant 21, 3324–3332 (2021). 80. S. R. Sharpton, N. A. Terrault, A. M. Posselt, Outcomes of sleeve gastrectomy in obese liver transplant candidates. Liver Transpl 25, 538–544 (2019). 81. D. Zamora-Valdes et al., Long-term outcomes of patients undergoing simultaneous liver transplantation and sleeve gastrectomy. Hepatology 68, 485–495 (2018). 82. M. C. Morris et al., Delayed sleeve gastrectomy following liver transplantation: A 5-year experience. Liver Transpl 25, 1673–1681 (2019). 83. V. Lopez-Lopez et al., Are we ready for bariatric surgery in a liver transplant program? A meta-analysis. Obes Surg 31, 1214–1222 (2021). 84. A. J. Sanyal et al., Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med 362, 1675–1685 (2010). 85. K. Cusi et al., Long-term pioglitazone treatment for patients with nonalcoholic steatohepatitis and prediabetes or type 2 diabetes mellitus: A randomized trial. Ann Intern Med 165, 305–315 (2016). 86. P. N. Newsome et al., A placebo-controlled trial of subcutaneous semaglutide in nonalcoholic steatohepatitis. N Engl J Med 384, 1113–1124 (2021). 87. D. M. Rubino et al., Effect of weekly subcutaneous semaglutide vs daily liraglutide on body weight in adults with overweight or obesity without diabetes: The STEP 8 randomized clinical trial. JAMA 327, 138–150 (2022). 88. R. A. Bhanji et al., Differing impact of sarcopenia and frailty in nonalcoholic steatohepatitis and alcoholic liver disease. Liver Transpl 25, 14–24 (2019). 89. S. S. Patel et al., The impact of coronary artery disease and statins on survival after liver transplantation. Liver Transpl 25, 1514–1523 (2019). 90. P. E. Frasco et al., Statin therapy and the incidence of thromboembolism and vascular events following liver transplantation. Liver Transpl 27, 1432–1442 (2021). 91. P. T. Campbell, L. B. VanWagner, Mind the gap: Statin underutilization and impact on mortality in liver transplant recipients. Liver Transpl 25, 1477–1479 (2019). 92. J. I. Lee, H. W. Lee, K. S. Lee, H. S. Lee, J. Y. Park, Effects of statin use on the development and progression of nonalcoholic fatty liver disease: A nationwide nested case-control study. Am J Gastroenterol 116, 116–124 (2021). 93. B. Zou, M. C. Odden, M. H. Nguyen, Statin use and reduced hepatocellular carcinoma risk in patients with non-alcoholic fatty liver disease. Clin Gastroenterol Hepatol, (2022). 94. F. M. Chang et al., Statins decrease the risk of decompensation in hepatitis B virus- and hepatitis C virusrelated cirrhosis: A population-based study. Hepatology 66, 896–907 (2017). 95. A. Mohanty, J. P. Tate, G. Garcia-Tsao, Statins are associated with a decreased risk of decompensation and death in veterans with hepatitis C-related compensated cirrhosis. Gastroenterology 150, 430–440.e431 (2016). 96. M. Bhat, K. Mara, R. Dierkhising, K. D. Watt, Gender, Race and disease etiology predict de novo malignancy risk after liver transplantation: Insights for future individualized cancer screening guidance. Transplantation 103, 91–100 (2019).

24 NAFLD IN LEAN INDIVIDUALS

24 NAFLD in Lean Individuals Donghee Kim and Vincent Wai-Sun Wong

CONTENTS 24.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 24.2 What Is Lean? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 24.3 Prevalence of NAFLD in Lean or Nonobese Individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226 24.3.1 Prevalence of Nonalcoholic Steatohepatitis (NASH) and Fibrosis in Lean Individuals . . . . . . . . . . . . . . . . . 227 24.4 Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.1 Visceral Adiposity and Sarcopenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.2 Other Metabolic Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.3 Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.4 Genetic Risk Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.4.1 PNPLA3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.4.2 TM6SF2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228 24.4.5 Gut Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 24.5 Clinical Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 24.6 Clinical Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 24.7 Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 24.8 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 24.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Key Points ■

NAFLD is strongly associated with obesity and the related metabolic disorders. Nonetheless, NAFLD may be present in 5–27% of patients with a relatively normal body mass index (BMI).

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Long thought to be an Asian phenomenon, NAFLD has now been reported in lean individuals of different ethnic backgrounds.

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Despite normal BMI, lean individuals with NAFLD tend to have visceral obesity and other metabolic risk factors. Genetic predisposition (e.g., PNPLA3 and TM6SF2 gene polymorphism) and gut microbiota dysbiosis also contribute to NAFLD development. More research is needed to defne which pathogenic mechanisms are specifc to the development of NAFLD in lean individuals.

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In general, lean individuals have less severe NAFLD. Data on disease progression are conficting, with a few studies suggesting that a subset of lean patients may be at a higher risk of liver-related morbidity and mortality in the long run than obese patients.

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Noninvasive tests of fbrosis such as simple fbrosis scores, specifc biomarkers and imaging techniques perform similarly in lean and obese patients and can be used for initial assessment.

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Lean patients with NAFLD also beneft from lifestyle intervention but can achieve improvements in liver fat at a smaller degree of weight reduction than obese patients. Few if any studies examined the effect of pharmacological treatment in lean patients with NASH. The latter should be addressed in future studies.

24.1 INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is strongly associated with obesity and metabolic syndrome.1 In particular, DOI: 10.1201/9781003386698-29

insulin resistance is almost universal in NAFLD patients even before the development of diabetes.2 Nonetheless, it is clear from numerous observational and epidemiological studies that a proportion of patients with NAFLD may have relatively normal body mass index (BMI). This is often referred to as “lean” or “nonobese” NAFLD. In the early 2000s, it was generally believed that this was an Asian phenomenon as most publications on this topic came from Asia, and Asians tended to have central obesity at a lower BMI.3 However, subsequent studies showed that the same could be seen in Western countries. In this chapter, we will frst discuss the defnitions of obesity and leanness and then review the epidemiology, pathogenesis, clinical features and outcomes of NAFLD in lean patients. Based on the knowledge, we discuss the implications on the assessment and treatment of this condition. 24.2 WHAT IS LEAN? According to the World Health Organization (WHO), BMI is a crude measure of overweight and obesity.4 BMI is calculated as body weight (kg) divided by body height (m) squared. Using BMI, people’s nutritional status can be classifed into underweight (BMI 40 kg/m2). People with BMI